In the present study, anaerobic co-digestion process was carried out with 23 mixed substrates proportion (MSP) of fruit waste (FRW), municipal wastewater treatment plant aerated biosolid (MPABS) and dairy effluent treatment plant returned biosolid (DPRBS). During co-digestion process, the effect of MSP on carbon/nitrogen (C/N) ratio and hydrogen production was investigated. The results revealed that MSP17 (70 FRW:20 MPABS:10 DPRBS) has yielded maximal hydrogen production of 295 mL with C/N ratio of 30, followed by MSP9 (70 FRW:30 DPRBS) exhibiting 253 mL of hydrogen production with C/N ratio of 29 and MSP2 (90 FRW:10 MPABS) attained 223 mL of hydrogen production with C/N ratio of 27. Then, SEM analysis of digested substrate sample was also performed in which flocs observed to be small and loose in structure in co-digested samples and intact form in non co-digested samples. Hence, this study results can be used for a sustainable approach by utilizing the FRW and biosolids for hydrogen production.