Crystallization-induced microscopic stress and its relaxation play a vital role in understanding crystallization behavior and mechanism. However, the real-time measurements for stress and its relaxation seem to be an unachievable task due to difficulties in simultaneous labeling, spatiotemporal discrimination, and continuous quantification. We designed a micron-sized fluorescent probe, whose fluorescence can respond to stress-induced environmental rigidity and whose three-dimensional (3D) flow can respond to stress relaxation. Using the as-prepared fluorescent probe, we established a versatile strategy to realize the real-time 3D imaging of stress and its relaxation in the crystallization process. The rigidity-responsive fluorescence clearly indicated the stress, while the 3D flow movement could quantify the stress relaxation. It is revealed that stress in spherulites increased dramatically as a result of the suppression of stress relaxation in polymer melts. The developed method provides a novel avenue to simultaneously detect stress and its relaxation in various semicrystalline polymers at the single-particle level. This success would achieve the microscopic ways to guide the development of advanced crystallization-dependent materials.