In this study, the usability of AChE immobilized nanospheres (NSs) was investigated to identify Imidan Phosmet and Vantex pesticides. Accordingly, (2AEPS-SalBr) coded nanosphere was synthesized in result of condensation of (Tris (2 aminoethyl)amine polymer and 5-bromine-2-hydroxy benzaldehyde. Then, new nanosphere coded 2AEPS-SalBr-Pd was synthesized using the template method. The structures of the synthesized nanospheres were characterized by the FT-IR, GPC, SEM/EDX and elemental analysis. Acetylcholinesterase (AChE) enzyme was immobilized to these nanospheres for the determination of Imidan and Vantex. The optimum conditions of the immobilized-AChE enzyme, the number of reuse, storage stability, and the kinetic parameters were determined. The immobilized AChE optimum pH was 8 for both supports, and the optimum temperature was 50 degrees C and 70 degrees C, respectively. Km and Vmax values were calculated as 1.443 mM and 0.251 mM/min for (2AEPS-SalBr)-AChE (pH 8, at 50 degrees C), and 0.680 mM and 0.157 mM/min for (2AEPS-SalBr-Pd)-AChE (pH 8, at 70 degrees C). Then the analyses of Imidan Phosmet and Vantex pesticides were carried out with immobilized AChE on nanospheres. As a result of the research, it was seen that Imidan Phosmet pesticide can be determined at a lower concentration than Vantex pesticide.