The fabrication of a convenient fluorescence-based immunoassay (FIA) for specific antigen attract increasing concern but remains a considerable challenge, especially the laborious synthesis of nanomaterials-antibody conjugates. Herein, we circumvent this drawback by introducing a specific fluorescence signal response procedure to commercially available alkaline phosphatase (ALP)-labeled conventional immunoassay for imidacloprid detection. In this FIA protocol, gold nanoclusters (AuNCs) can anchor on the surface of two-dimensional cobalt oxyhydroxide (CoOOH) nanoflakes to form nanocomposite, resulting in remarkable decrease of fluorescence intensity. The quenching effects can be effectively reversed by introducing ascorbic acid that can trigger the decomposition of CoOOH nanoflakes. Notably, the corresponding fluorescence response induced by ascorbic acid was related to ALP activities labeled on antibody. After competitive immunoreaction, the ALP-labeled antibodies were bounded to immobilized antigen, which can regulate the fluorescence change. Utilizing fluorescence switching of system, the 50% inhibition concentration (IC50) value of FIA toward imidacloprid was obtained at 1.3 ng mL^-1which was 60-fold-of-magnitude more sensitive than that of conventional ELISA (86.4 ng mL^-1). This FIA protocol not only develops new prospects for pesticide detection, but also opens up potent strategy of fluorogenic immunoassay.