Hollow carbon spheres-ionic liquid 1-butyl-3-methyl imidazole phosphotungstate (HCS-[Bmim]3PW12O40) was prepared to modify glassy carbon （GC） electrode. Acetylcholinesterase (AChE) was immobilized on the electrode surface by cross-linking reaction to obtain AChE/HCS-[Bmim]3PW12O40/GC sensor. Under the optimal condition, the organophosphorus pesticides, monocrotophos and carbofuran, were detected by the biosensor. The linear range of monocrotophos was 8.00×10-10~8.00×10-6 g/L, and that of carbofuran 1.00×10-10~1.00×10-7 g/L, with the detection limits of 7.58×10-11 g/L and 6.76×10-11 g/L, respectively, calculated by inhibition rate 10%. The biosensor exhibited high sensitivity and acceptable stability, and could be a promising tool for analysis of organophosphorus pesticides.