In this study, hydrogen (H2) production from water hyacinth (WH) was enhanced by the integration of the ultrasonic-assisted alkaline (UAA) pretreatment, dark fermentation (DF), and microbial electrolysis cell (MEC). The results showed that UAA pretreatment improved around 350% in H2 production in the DF stage and nearly 400% in the whole process compared to un-pretreated. The H2 yield in the DF stage reached the maximum value of 110.4 mL/g-VS at a WH concentration of 20 g-TS/L. However, high concentrations of co-produced soluble metabolite products (SMPs) and suspended solid in DF effluent adversely affected the efficiency of the MEC stage. Consequently, a WH concentration of 5 g-TS/L was optimal for the UAA-DF-MEC process that achieved the highest H2 yield of 565.8 mL/g-VS. It suggests that other auxiliary processes (e.g., dilution, centrifugation, effective methanogen inhibition, etc.) need to be developed to further improve the H2 production from WH via the UAA-DF-MEC process.