Currently, pesticide residues increasingly lead to health problems. Acetylcholinesterase enzyme (AChE) plays a role in detoxification processes due to their ability to scavenge organophosphates. Thus, this enzyme has been selected for the detection of pesticides. Herein, this study describes the preparation and application of appropriate new immobilized spheres that could be used for the detection of organophosphorus pesticide residues. That is a simple colorimetric enzymatic assay for the practical discovery of N-(Mercaptomethyl) phthalimide S-(O, O-dimethylphosphorodithioate) from organophosphorus pesticides (OPs) widely used in the treatment of sugar beet, apple, nut, corn, and tobacco. The process is based on the immobilization of acetylcholinesterase onto tryptophan FMPS-Trp, (FMPS-Trp)Pd(II) and (FMPS-Trp)Pt(II) functionalized spheres. Spectroscopic and microscopic techniques were used in the characterization of the spheres where scanning electron microscopy (SEM) is an essential tool. The (FMPS-Trp)Pt(II)-AChE showed a good affinity to acetylthiocholine chloride (ATCl) and was found to have the ability to catalyze the hydrolysis of ATCl with an apparent Michaelis–Menten constant value of 288 mM. The developed colorimetric process showed good qualitative analytical performance for Phosmet (OPs) detection as low as 10−7 M.