Various bioactive substances are simultaneously involved in physiological processes, and research on the synergistic effect of them can promote the study of pathological mechanisms. To achieve this purpose, several small-molecule double-locked logic gate fluorescence probes have been developed recently. They overcome many shortcomings of the traditional "single-signal" fluorescent probes, with fluorescence that can be activated by two analytes of interest order-independently or order-dependently with one output. In this review, we summarize recently published small-molecule double-locked logic gate probes for the optical detection of two bioactive substances in living systems. We envision that this review will attract significant attention from researchers to exploit more powerful functional double-locked logic gate probes.