In response to current energy challenges, anaerobic digestion (AD) is emerging as a viable and sustainable alternative to conventional fossil fuels. Phase separation evolves to mitigate process instability observed in single-stage AD, offering enhanced process stability and efficiency for biogas production. This study aims to comprehensively compare phase-separated and single-phase AD systems while incorporating cattle manure and fruit and vegetable waste (FVW) co-digestion to enhance process stability and biogas production. The two-stage and single-stage continuous AD systems were analyzed at an organic loading rate (OLR) of 3 gVSL-1day-1 with a hydraulic retention time (HRT) of 10 days and three retention periods (30 days) under uncontrolled environmental conditions (20-35ºC), which may fluctuate with seasonal changes, and controlled temperature (37ºC). The FVW and cattle manure were co-digested in a 1:1 ratio of volatile solids (VS) as a substrate. The two-stage AD system operated at 37ºC and exhibited the highest biogas yield (0.25 NL/gVS) at a steady state as compared to single-stage AD, including 0.113 NL/gVS, 0.178 NL/gVS, and 0.201 NL/gVS at environmental temperature (20-35ºC), controlled temperature (37ºC), and two-phase AD at environmental temperature (20-35ºC), respectively. The one-way analysis of variance (ANOVA) test revealed a significant effect of the controlled temperature and phase separation on biogas production, F(3, 0) = 0.987, p<0.001. Additionally, the total reduction in COD in the final effluent was notably higher in the two-stage anaerobic digester at 37ºC, showing an 85% reduction rate compared to single-stage anaerobic digesters with a mean difference of -6.51 (95% CI [-9.50, -3.52]) and SD = 1.88, t(3) = -6.93, p = 0.006, two-tailed. These results highlight the significant biogas productivity, improved process stability, and better effluent quality achieved through phase separation of the anaerobic digester at 37ºC. The estimated biogas potential from two-stage anaerobic co-digestion of cattle manure and FVW is 21.37 million m³/day in Pakistan, representing significant prospects for implementing this technology as a renewable energy source.