The effective enhancement of short-chain fatty acid co-metabolic methane production is a research hotspot. N-acyl homoserine lactones (AHLs) are effective means regulating anaerobic digestion behaviors. However, what factors influence the secretion of endogenous signaling molecules has not been studied. We established a propionic acid co-metabolic methane production system to examined how ecological factors affect AHLs secretion. We found that propionic acid concentration negatively correlated with C4-HSL secretion (-0.71 for R). Ammonia nitrogen positively correlated with C4-HSL secretion at low 100-1000 mg/L (0.89 for R), and negatively correlated at 1000-6000 mg/L (-0.95 for R). While pH positively correlated with C6-HSL (0.76 for R). Syntrophobacter, Syntrophorhabdus, and Methanospirillum significantly affected C4-HSL secretion. We also demonstrated a significant correlation between signaling molecules, syntrophic bacteria, hydrogenotrophic methanogens, and maximum methane production. Our study provided a new understanding for the future research of using AHLs to regulate the metabolism of syntrophic bacteria for methane production.