The quantitative determination of major enzyme activities in postharvest fruit is a necessary biochemical experiment to investigate the physiological and metabolic mechanisms of fruit ripeness, senescence, and stress responses. However, it is necessary to choose appropriate methods to stably extract and determine target analytes from complex plant matrices due to the dynamic changes in enzyme activities of postharvest fruit during storage. Here, we outline sensitive and accurate methods for measuring fruit enzyme activities associated with key physiological processes including respiration, reactive oxygen species scavenging, membrane lipid degradation, energy synthesis, cell walls degradation, and disease resistance. This manuscript aims to provide guidance for future research in postharvest biology.