Microbial Electrolysis Cell (MEC) is a promising technology enabling the sustainable production of hydrogen as energy alternative from renewable sources, such as wastewater, with low energy input. Currently, the applications of MECs are still limited due to the relatively low hydrogen production rate. MEC optimization still focused on the reactor construction design and external factors while the optimization of MEC from internal factor, which is electron transfer from microorganisms in the system, is still needed. This study focused on investigating the influence of anodic biofilm formation time to biohydrogen production in MEC system. The research works by Single-Chamber MEC configuration with optimum operating condition including the addition of denitrifier bacteria. The anodic biofilm formation is adjusted by giving various biofilm formation time prior to MEC operation. Hydrogen concentrations produced at the cathode are measured through Gas Chromatography and anodic biofilm formation at the anode is measured through SEM analysis. The study suggested that hydrogen yield for reactor with enriched biofilm could be higher in comparison to unenriched reactor, as electron used for hydrogen production transferred effectively with the help of electrochemical linkage of anodic biofilm. Experimental results showed H2 yield with five days incubation prior to MEC operation producing up to 70.69% compared to the control.