In this work, a simple, sensitive and selective colorimetric method was established for the detection of profenofos. Firstly, novel flower-shaped AuPtRh trimetallic nanospheres were synthesized a simple one-pot method, and had outstanding peroxidase catalytic activity. AuPtRh nanospheres with a great specific surface area were linked with an aptamer Au-S and Pt-S bonds to specifically recognize profenofos. A graphene oxide grafted stainless-steel mesh (SSM-GO) was prepared to be a carrier and the aptamer-AuPtRh was nonspecifically adsorbed on the surface of SSM-GO, which was to be the capture probe for the detection of profenofos in real samples. They were characterized and confirmed by transmission electron microscopy, atomic force microscopy, . Through the investigation of the catalytic performance on the basis of the Michaelis equation, the of AuPtRh nanospheres was 22.27 × 10 M s, and was 0.6632 mM, which indicated that the affinity of AuPtRh nanospheres was relatively higher than that of horseradish peroxidase and Au NPs. In the presence of profenofos, the aptamer-AuPtRh would specifically combine with profenofos, which would further detach from SSM-GO. Then, it was introduced into the 3,3',5,5'-tetramethylbenzidine/HO (TMB/HO) system to form blue oxTMB. The linear range of this colorimetric biosensor was 1-300 ng L and the limit of detection was 0.725 ng L. It also had good recovery and anti-interference ability in real samples, which provided a new strategy for the rapid detection of pesticides.