In this work, with the aim of developing effective molecular probes and investigating the structure-reactivity correlation, a short series of phenothiazine-based fluorescent probes are designed for the detection of ClO- with differing electron push-pull groups. Sensing experiment results and single-crystal X-ray analysis with the aid of time-dependent DFT (TD-DFT) calculations reveal that substituting groups with increasing electron-withdrawing ability can increase the dihedral angle of the phenothiazine moiety and reduce the gap energy of the probes, leading to enhanced reactivity toward ClO-. Both PT1 and PT2 show two-color switching upon detection of ClO-. PT1, with the strong electron-donating group thiophene, shows a fluorescence color switch from salmon to blue. PT2, with a medium electron-donating/accepting group benzothiazole, shows a fluorescence color switch from red to green. However, both PT1 and PT2 show almost no response to ONOO-. Through the introduction of strong electron-withdrawing ketone combined with a cyano group, PT3 shows a cyan emission upon detection of ClO- and weak red emission upon detection of ONOO-. HRMS and (HNMR)-H-1 results confirm that PT1 and PT2 have the same sensing mode, in which the divalent sulfur of phenothiazine can be oxidized to sulfoxide by ClO-. Upon reaction with ClO-, PT3 experiences two-step reactions. It is first oxidized into the sulfone structure by ClO-, and then transformed into sulfoxide phenothiazine aldehyde. Upon encountering ONOO-, PT3 changes into an aldehyde structure and some nonfluorescent byproducts. Owing to their special selectivity and high sensitivity, PT1 and PT2 are applied to image the endogenous ClO- in macrophage cells and zebrafish larvae. This study is expected to provide useful guidelines for probe design for various applications.