In the physiological and pathological processes, evaluating the role of peroxynitrite (ONOO-) in living cells with high sensitivity and selectivity is a significant challenge. In this study, a highly sensitive surface-enhanced Raman spectroscopy sensor (SERS) was proposed using 3-methoxyphenylborate pinacol ester (MAPE) modified gold nanoflowers (Au NFs/MAPE) for the detection of ONOO- in living cells. The principle of the nanosensor is based on ONOO- which can oxidize aryl boric acid on Au NFs to release phenol, resulting in changes in the SERS spectrum of the sensors. The results show that the SERS sensors have high selectivity and excellent sensitivity for ONOO- in the presence of other interfering ions. The detection limit of the sensors is as low as 0.48 μM, which can meet the needs in biological systems. In addition, the SERS sensors have long-term stability and high biocompatibility. More importantly, this sensor can successfully monitor active biological samples and detect the ONOO- content generated during an oxidative stress reaction, creating a new method for studying cell biochemistry.