The hydrogen embrittlement (HE) phenomenon in titanium alloys poses a critical challenge to their application in deep-sea environments and hydrogen energy fields. Studying the influence of the microstructure of titanium alloys on the formation mechanism of hydrides is the key to regulating the hydrogen embrittlement resistance of the alloys. The diffusion behavior of hydrogen in lamellar and bi-modal TC4 alloys is investigated through electrochemical hydrogenation combined with multi-scale characterization techniques. The results show that after electrochemical hydrogen charging, the diffusion surfaces of lamellar and bi-modal samples present a gradient distribution of hydrogen concentration, and the thickness of the hydrogen diffusion layer of two microstructure is similar. The volume fraction of hydride in the diffusion surface of the lamellar sample is larger, hydrides preferentially form at the α/β interface and grow in the form of twin pairs into the α phase. In the case of the bi-modal structure sample, due to the relatively large equiaxed α grain size, hydrides cannot fill the entire α grain. Different hydride variants alternate in nucleation and growth near the α/β interface. Transmission electron microscopy analysis results indicate that the hydrogenation nucleation in both microstructure samples presents a multi-level structural transformation mechanism regulated by stacking faults.