The investigation on the interaction mechanism between pesticide pyriproxyfen (PPF) and serum albumin has great implications in clinical detection, gene mutation and pathological analysis of pesticide poisoning. In this paper, the binding behavior of PPF with bovine serum albumin (BSA) have been assessed through various spectroscopic techniques combined with computer simulation. The findings confirmed that PPF quenched the endogenous fluorescence of BSA in the means of static quenching and formed the stable PPF‐BSA complex with the stoichiometry of 1:1. The affinity of PPF on BSA was moderate due to its binding constant of 4.15×103 M−1 (298 K). It was confirmed from replacement experiments and molecular docking that PPF bound preferentially onto the Site I region of BSA. The findings from thermodynamic parameter analysis and the replacement experiments of ANS and sucrose confirmed the driving‐forces for forming PPF‐BSA complex was hydrogen bonding, van der Waals and hydrophobic interactions. Meantime, it was also confirmed from synchronous fluorescence and FT‐IR spectra that the hydrophilicity surrounding Trp residues and α‐helix of BSA declined due to binding with PPF. And, it is confirmed from in silico finding the dipole moment, atomic charge distribution, molecular conformation, and frontier orbital of PPF also significantly altered after binding with BSA.