〔Abstract〕 Objective To construct a high-throughput in vitro method for the detection of botulinum toxin A enzyme activity by using fluorescence resonance energy transfer(FRET). Methods Recombinant expression plasmid based on double fluorescent labeled substrate was constructed to recognize botulinum toxin type A only, and the constructed plasmid was transferred into the E.coli expression system for expression. The expressed recombinant protein of fluorescent labeled substrate was purified, dialyzed and stored for standby; The activity of the recombinant protein was detected by digestion of botulinum toxin type A light chain(ALc); The conditions of this detection method were optimized; The enzyme kinetic parameters Kmand Kcatwere determined by cutting the fluorescent labeled substrate with ALc. Results The recombinant expression plasmid was successfully constructed. After being expressed in the E. coli expression system, the target band appeared obviously. The purity of the purified recombinant protein was about 90%. The recombinant protein was named CYA. CYA was identified by enzyme digestion of ALc and Botulinum toxin type B light chain(BLc). The results showed that CYA could only be digested by ALc to produce two protein fragments that were consistent with expectations, but could not be digested by BLc. By optimizing the conditions based on FRET substrate, it was obtained that the filter sensitivity was set between 65-110; The real-time dynamic detection interval was 2 min/time, the dynamic detection time was 30-120 min, and the appropriate concentration range of substrate CYA was 0.5-32 μmol/L. The ratio of the time change of CYA at any time under the action of ALc enzyme to the fluorescence value 528 and 485 was plotted to be about 0.5 at the minimum and 0.9 at the maximum. The enzyme kinetic parameters determined that the value of ALC cleaving CYA Kcatwas(5±0.4) s-1and Kmwas(2.33±0.21) μmol/L. Conclusion A high-throughput in vitro method for the detection of botulinum toxin type A activity based on FRET technology is successfully constructed.