Abstract: The value of ecological service function (including crop production, atmosphere regulation, water storage, nutrient cycle and soil conservation) of agro-ecosystems under typical ricewheat rotation in Jiangsu Province was comprehensively evaluated using three evaluation methods (market value, replacement valuation and shadow price). Results show that rice-wheat rotation ecosystem provides an average overall service value of 45 998.08 yuan·hm^-2·a^-1, in which environment regulation function value is 26 458.86 yuan·hm^-2·a^-1; 1.35 times that of direct service value of crop production. That means 1.00 yuan cost of rice-wheat rotation ecosystem provides an environmental benefit value of 3.58 yuan. Air regulation being the dominant constraint accounts for 62.20% environment regulation service value. The study reveals the significance of non-marketed ecological service of rice-wheat rotation agro-ecosystem, and that government should pay much attention on environment regulation function of agro-ecosystems in making decisions on land utilization.