Abstract: Ratooning rice has the advantages of saving production costs and improving grain yield. However, there are less reports on the effect of the combination of nitrogen fertilizer and organic materials on the soil in the ratooning rice mode. This study compared the effects of different nitrogen fertilizer combinations with organic materials on the soil properties of ratooning rice paddy fields to provide a reference for the sustainable development of ratooning rice models. A two-year (2020–2021) single-factor experiment was conducted in Jingzhou, Hubei, China. The experiment included five base fertilizer treatments: no nitrogen fertilizer (N0), base fertilizer nitrogen from conventional urea (CK), 50% base fertilizer nitrogen from conventional urea and 50% from slow-release urea (T1), biochar (T2), or animal manure (T3). The fertilization mode of T2 was only conducted in 2020, which was the same as that of CK in 2021; The fertilization modes of the other treatments were the same for both years. Compared with N0, the bulk density (BD) in the 0–20 cm soil layer at the heading stage of the main season rice and at the heading stage of the ratooning season rice decreased by 3.92%–6.15% in CK and by 4.38%–6.74% in T1, whereas the BD at the 0–40 cm soil layer during the whole growth period decreased by 9.82%–17.87% in T2 and by 9.48%–14.21% in T3. The order of soil pH in 2020 was T2>T3>N0>T1>CK. Compared with CK, pH in 2020 increased by 0.51−0.68 in T2. The order of soil pH in 2021 was T3>T2>N0>T1>CK. Compared with CK, the pH in 2021 increased by 0.14−0.32 in T3. The content of soil organic carbon (SOC) and total nitrogen (TN) in the 0–20 cm and 20–40 cm soil layers were T2>T3>T1>CK>N0. Compared with N0, other treatments increased the content of SOC and TN in the 0−20 cm soil layer by 4.79%−29.12% and 11.36%−28.49%, respectively; and they increased the contents of SOC and TN in the 20−40 cm soil layer by 5.43%−30.79% and 6.08%−20.02%, respectively. The contents of NH₄⁺ and NO₃⁻ at the tillering and heading stages of the main season rice and the heading stage of the ratooning season rice were the highest under the CK, T1, and T3 treatments. Compared with N0, the contents of NH₄⁺ and NO₃⁻ at the tillering stage of the main season rice increased by 131.26% and 153.59% under the CK treatment, respectively; the contents NH₄⁺ and NO₃⁻ at the heading stage of the main season rice increased by 217.15% and 153.91%, respectively, under the T1 treatment; and the contents of NH₄⁺ and NO₃⁻ at the heading stage of ratooning season rice increased by 246.76% and 126.70%, respectively, under the T3 treatment. Microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), and urease (UR) activity in T2 and T3 were higher than in the other treatments. Compared with N0, MBC content increased by 18.29%−45.18% in T2 and 21.46%−46.10% in T3, MBN content increased by 49.25%−140.37% in T2 and 59.62%−142.57% in T3, and UR activity increased by 31.45%−225.04% in T2 and 60.83%−246.65% in T3. The β-glucosidase (BG) activity was the highest in the T3 treatment. Compared with N0, the BG activity increased by 21.26%−44.87% under the T3 treatment. A comparative analysis showed that the effect of animal manure on reducing BD and improving SOC and TN was similar to that of biochar, and its effect on improving inorganic nitrogen, microbial biomass, and soil enzyme activity was better than that of biochar. Therefore, animal manure and chemical fertilizers should be used as base fertilizers, and animal manure should replace 50% of the chemical fertilizer nitrogen.