Ratiometric fluorescent probes could effectively offset the changes of the autofluorescence and compartmental localization. FRET, ICT, etc. are the common strategies to design probes for biosensing, but these strategies have some deficiencies. Here, we proposed a new design strategy based on π-conjugation modulation, giving two different emission bands in the absence and presence of the target. The new fluorescence probe named Rhod-DCM-B was rationally designed and synthesized, which displayed a fluorescence emission peak at 670 nm because the electron cloud focuses on the conjugated DCM unit. With the addition of ONOO-, the fluorescence emission at 570 nm increased, accompanied by the decrease of fluorescence emission at 670 nm, showing a ratiometric signal change attributed to the opened spirane structure making the electron cloud concentrated on the xanthene core. The mechanism is well confirmed by MS and DFT calculations. Rhod-DCM-B exhibited outstanding sensitivity and excellent selectivity toward ONOO-. Moreover, Rhod-DCM-B was effectively employed to determine endogenous and exogenous ONOO- in living cells. As a marker for inflammation and drug-induced liver injury (DILI) process, ONOO- in vivo was successfully monitored by Rhod-DCM-B and presented a dramatic ratiometric response.