By virtue of their unique features, including simplicity, speed and portability, point-of-care testing (POCT) is critical for biochemical analysis in resource-limited environments. However, a single-signal readout cannot fulfil the requirements of accurate quantitative analysis due to interference from environmental factors. Hence, establishing a convenient multimodal assay method based on the concept of POCT detection is urgently desired. Herein, a novel multimode (i.e., photothermal, electrochemical, UV-Vis absorption and fluorescence) sensing strategy based on oxygen-vacancy-rich MnO2 nanosheets decorated with methylene blue (MB) is unveiled for multifunctional detection of ascorbic acid (AA). In this multimode nanosensor (MnO2@MB NSs), MnO2 nanosheets act as a photothermal agent and carrier for MB, showing an ultrasensitive response to AA, which enables photothermal signals turn “on” to “off”, and can be monitored by a thermometer. In the meantime, large amounts of MB are released to the detection solution, resulting in the absorbance, fluorescence and electrochemistry signals turning “off” to “on”. Thus, this nanosensor enables four-signal determination of AA in a supersensitive manner and exhibits a more reliable analysis result. Furthermore, MnO2@MB NSs can be even used to achieve point-of-care testing, greatly improving their application scope. Our strategy opens up a new avenue for the exploration of multistimulus-responsive nanosensors for convenient, stable, and accurate analysis of AA.