In this paper, Prussian blue nanoparticles (PBNPs) and colloidal gold (AuNPs) were synthesized by solvothermal method and trisodium citrate reduction method respectively, and two lateral flow immunoassay (LFIA) methods for the detection of ofloxacin (OFL) in fish were established. The larger particle size and superior optical properties of PBNPs make the sensitivity of PBNPs-LFIA higher than that of AuNPs-LFIA. In the process of establishing the assay, we found that the amount of antibody required for PBNPs-LFIA is 1/3 of that for AuNPs-LFIA, and the amount of probe is 1/2 of that for AuNPs-LFIA, suggesting that PBNPs-LFIA is able to save the amount of antibody and greatly reduce the cost of the assay.The detection limit of PBNPs-LFIA for OFL was 0.064 ng mL−1, which was 2.3 times lower than that of AuNPs-LFIA, and the visual detection limit and elimination value of PBNPs-LFIA were also lower than that of AuNPs-LFIA.In addition, the recoveries of two established LFIAs for the determination of OFL in fish were 84.8 %-116.3 %, and the coefficient of variation was less than 10 %. Therefore, PBNPs are a potential LFIA probe marker.