In recent years, environmental endocrine disruptors have attracted more and more attention. Traditional detection methods are time-consuming, complex and expensive, which means the development of fast response, high sensitivity and low cost detection method is of vital importance. Herein we described a planar waveguide-based array evanescent wave fluorescent biosensor. Based on the simulation and optimization of the optical parameters of the system, it allows the measurements of up to twenty-four sensing spots simultaneously, rapidly and highly sensitive. By using the indirect competitive immunoassay, the detection conditions of the array evanescent wave fluorescent biosensor were optimized and established for the environmental endocrine disruptor of estradiol. The experimental results show that the system has the highest sensitivity at 61.8°. The linear response ranged from 0.08~2.52 μg·L-1 has the detection limit of 0.05 μg·L-1 and the 50% inhibition concentration (IC50) of 0.46 μg·L-1. One test cycle was completed within 20 min. The recoveries of estradiol in all sorts of wastewater effluents ranged from 84% to 120% has relative standard deviation values of less than 15%, confirming the application potential in the measurement of estradiol in reality.