Replacing virgin mining with urban mining is an important step to achieve resources recycling and carbon emission reduction. Bio-cyanidation is an environmentally-friendly technology to recover precious metals from waste printed circuit boards, a type of typical urban minerals. However, the limited cyanide-producing ability and the risk of cyanide containing solutions restricts its industrial applications. Investigating the regulation mechanism of cyanide production and conversion helps solve the current dilemma. In this experiment, the concentration of cyanide produced by Pseudomonas fluorescens increased first and then decrease, involving two processes of cyanide production and conversion. The quorum sensing signal concentration was 1.34 µg/L, 1.96 µg/L, and 2.32 µg/L at 12 h, 18 h, and 24 h, respectively. Meanwhile, the transcriptome profiling revealed that the differentially expressed genes were enriched in the pathway of quorum sensing. Combined the two results, the quorum sensing was inferred as the drive of cyanide production. Unused cyanide was converted into β-cyanoalanine by β-cyanoalanine synthase, of which the concentration reached the lowest at the cyanide concentration peak. The findings help regulate the cyanide-production and conversion in bio-cyanidation process and contributes to the recovery of precious metals from waste printed circuit boards.