The tempering process is applied in marine 10Ni5CrMoV steel to study microstructure, and mechanical properties by multi‐scale characterizations, strain hardening behavior, and cryogenic toughening mechanism are further investigated. As the tempering temperature increases from 590 to 630 °C, the dislocation density decreases by up to 25% and the austenite volume fraction decreases by up to 35%. Additionally, the morphology of austenite changes from strip to bulk, which weakened the pinning effect on the laths, leading to a coarsened martensite and an increase in the equivalent grain size. Based on the modified Crussard–Jaoul analysis, the specimens at different tempering temperatures exhibit a single‐stage strain hardening behavior during plastic deformation. The increase in strength is attributed to the continuous transformation‐induced plasticity effect and the increasing dislocation density. Furthermore, high austenite volume fraction and the proportion of grain boundary misorientation above 45° promote the release of stress concentration and hinder the propagation of cracks. This results in an increase in the ductile–brittle transition temperature (DBTT) of the specimens from −105 to −135 °C. At a tempering temperature of 610 °C, the specimen demonstrates an outstanding balance between yield strength (868 MPa) and cryogenic toughness (DBTT of −135 °C).