Crystallization of metallic glass is not only affected by undercooling but also influenced by microstructure. However, the role of structure in the crystallization is still ambiguous. In this study, nanocalorimetry with a rapid quenching rate was used to obtain the critical cooling rates suppressing nucleation and crystal growth, realizing the in-situ preparation of the amorphous phase. Based on the nuclei-free metallic glass, annealing at Tg was performed to tune the microstructure. With a reheating analysis, the consecutive occurrence of relaxation, nucleation, and crystal growth during annealing was distinguished based on the evolution of crystallization heat. Moreover, the effect of annealing and the resulting microstructure evolution on the reheating crystallization was demonstrated. With the formation of nuclei, the stability of the undercooled melt deteriorated, causing the reduction of activation energy (Ea). When crystals formed during annealing, the confined residual amorphous phase was dominant for crystallization, and a higher Ea was observed.