3-Phenoxybenzoic acid（3-PBA），as the most common metabolite of pyrethroid pesticides，is typically employed as the risk indicator for pyrethroid pesticides exposure. Antibody-based immunoassay has the advantages of rapidity，high sensitivity and easy operation，which is considered as a promising tool for the risk exposure assessment on hazardous chemicals in environment，food stuffs and human body fluid samples. Nanobody（Nbs） is the variable domain of heavy chain-only antibody（VHH） derived from heavy chain antibodies（HCAbs） of camelidae or new antigen receptor（NAR） of shark，which are naturally lack of light chain. Unlike conventional antibodies，Nbs have high binding affinity with only a single domain，avoiding the complex combination of heavy and light chains during recombinant expression，which would sometime lead to the low solubility and stability of genetically engineered antibody fragments. Therefore，Nbs are easily expressed in soluble form，and have similar affinity with its natural structure. And Nbs have been reported to have excellent thermostability and organic solvent tolerance，which is benefit to simplify the pretreatment step. Thus，Nbs are considered as an ideal tool for developing rapid immunoassays. In this work，with the aim to develop a sensitive immunoassay for monitoring the metabolite of pyrethroid pesticides 3-PBA in water and human urine samples，the biotinylated 3-PBA Nbs using biotin N-hydroxysuccinimide ester（biotin－NHS） were prepared. To further amplify the signal of immunoassay，a polymeric horseradishperoxidase conjugated streptavidin（polyHRP－SA） was also introduced to develop a biotin－avidin system based indirect competitive enzyme-linked immunosorbent assay（icELISA）. The assay conditions were carefully optimized as follows：the coating antigen concentration was 125 ng/mL，the dilution ratio of biotinylated 3-PBA to Nbs was 1∶2 000，PBST with an ion concentration of 40 mmol/L，a pH 6.4 and a Tween－20 concentration of 0.005% （by volume） was optimal working buffer system，the dilution ratio of polyHRP－SA was 1∶4 000. The developed icELISA showed a half maximal inhibitory concentration（IC,50,） of 1.7 ng/mL，a linear range of 0.37－7.4 ng/mL and a limit of detection（LOD） of 0.15 ng/mL. The proposed Nbs-based icELISA was applied in the detection of 3-PBA in water and human urine samples. For water samples such as lake water， reservoir water，tap water and drinking water，the matrix effects could be easily eliminated by simply filtering and then used for icELISA without further dilution，while for the human urine samples，high temperature acid hydrolysis and solid phase extraction（SPE） purification are benefit to reduce the matrix effects. The spiked recoveries for human urine and water samples were in the range of 87.0%－127% and 78.0%－113%，respectively，with relative standard deviations（RSD） not more than 10%. Results indicated that the proposed method was sensitive and easy to operate，which could be used as an ideal tool for screening 3-PBA residues in environmental and biological samples.