InP/ZnS quantum dot (QD)-based fluorescent probe for directly sensitive and selective detection of horseradish peroxidase (HRP) was reported herein. Fluorescence of InP/ZnS QDs was statically quenched by HRP, due to the ground state complex formation of InP/ZnS QDs with HRP. Such ground state complex formation between InP/ZnS QDs and HRP reduced both the α-helix content and the melting temperature of HRP. Several key factors including InP/ZnS QDs concentration, buffer pH value, ionic strength, reaction temperature, and reaction time those affected the analytical performance of InP/ZnS QDs in HRP determination were investigated thoroughly. Under the optimal conditions, fluorescence intensity of InP/ZnS QDs was linearly decreased with the increasing of HRP concentration during the range of 1.0 × 10 M ∼ 3.0 × 10 M (0.01 U ml ∼ 0.3 U ml) with the detection limit as low as 1.2 × 10 M (1.2 mU ml). The present method showed excellent selectivity for HRP over some amino acids, nucleotides, and common proteins. This method was utilized to detect HRP in synthetic samples successfully.