Abstract: In order to conduct the visualized research of cellular pyruvic acid(PA), several rhodamine-based probes were designed to react with PA by a Schiff-base reaction in aqueous media and exhibit strong fluorescence emission and a pink color owing to the ring-opening of the corresponding spirolactam. The results showed that R6G-NH₂ had a highest sensitive response to PA with a linearly relationship between the enhancement of fluorescence intensity at 565 nm and the concentration of PA from 0. 2 μmol/L to 120 μmol/L. According to the principle of 3σ, a limit of detection was obtained as low as 0. 1 μmol/L. Furthermore, the response of R6G-NH₂ to PA was hardly affected by conventional biomolecules and ionic species. Therefore, R6G-NH₂ was successfully used in fluorescence imaging of exogenous PA in non-small cell lung cancer cell A549 and renal epithelial cell 293T, and endogenous PA induced by bisphenol A in epithelial cell 293T.