Wastewater-based epidemiology has some advantages in assessing chronic diseases with high prevalence, but selecting appropriate biomarkers is difficult. The aim of this study was to screen biomarkers in wastewater to accurately assess the prevalence of hypertension (ten drugs), diabetes (eight drugs), and gout (four drugs). The concentrations of biomarkers in the influent from 37 wastewater treatment plants in 34 Chinese cities were assessed using LC/MS−MS combined with solid-phase extraction. Biomarkers with excretion rates above 5% and high stability (>80%) and detection frequency (>80%) were selected to evaluate the prevalence. Sartans, hydrochlorothiazide, sotalol, and nornifedipine have been identified as biomarkers for hypertension; metformin and glibenclamide for diabetes; and oxypurinol and febuxostat for gout. The prevalence rates of hypertension, diabetes, and gout in China assessed using these biomarkers were 26% (95CI %: 18.8−36.5), 9.58% (95CI %: 6.62−14.2), and 5.95% (95CI %: 4.22−6.74), respectively, which were consistent with government survey data. We found a cooccurrence of hypertension, diabetes, and gout prevalence, and they were associated with unhealthy lifestyle, economics, and medical level. The results suggested that wastewater-based epidemiology is an effective approach to accurately assess disease prevalence by selecting multiple biomarkers, providing valuable information for public health.