Acetylcholinesterase (AChE) has been widely applied on the enzyme inhibition-based detection of organophosphate pesticides (OPs). To improve the sensitivity of fluorometric OPs assay, great efforts were made to change the fluorometric probes or analytical strategies rather than improve the sensitivity of AChE towards OPs. In this work, AChE wild-type (WT) and mutants (E69Y and E69Y/F330L) from Drosophila were successfully displayed on the surface of yeast through a-agglutinin-mediated microbial surface display system. The location of AChE on yeast surface was confirmed by immunofluorescence analysis. Further, a fluorescence OPs detection method was developed by combining yeast surface-displayed AChE mutants and protein-directed electronegative fluorescent gold nanoclusters (Au NCs). Yeast surface-displayed AChE can catalyze the hydrolysis of acetylthiocholine to produce thiocholine. The electropositive thiocholine can not only bind with AuNCs by Au-S bond but also absorb Au NCs by the electrostatic interaction, leading to the aggregation of AuNCs and corresponding fluorescence quenching. When AChE was incubated with paraoxon, a typical model of OPs, the activity of AChE was inhibited and the thiocholine-induced aggregation of AuNCs was reduced. The fluorescence assay based on Au NCs and yest-AChE-E69Y/F330L exhibited the ultra-sensitivity for ultra-trace OPs and 2-6 orders of magnitude lower detection limit (3.3 × 10 M) than those of AChE-WT-based method and other reported methods. In addition, the proposed method showed excellent reliability for the real samples assay. This work would provide an alternative strategy for the improvement of bio-analysis at its source.