Lead halide perovskite nanocrystals (NCs) exhibit excellent optoelectronic performance and have drawn great interests in the fields of biological imaging and sensing. However, the poor stability of CsPbX₃ (X = Cl, Br, I) in water is still a challenge to hinder their practical applications. In this work, a facile strategy has been developed for aqueous synthesis of CsPbX₃ nanocrystals, in which L-glutamic acid (L-Glu) has been used to replace oleic acid in the synthetic process. Benefiting from the synergic effects of L-Glu and oleylamine (OAm), CsPbBr₃ nanocrystals (L-Glu/OAm-CsPbBr₃ NCs) with high water stability have been directly prepared under a mild condition at room temperature in water, facilitated by the process of crystal phase transformation from Cs₄PbBr₆ to CsPbBr₃. L-Glu/OAm-CsPbBr₃ NCs exhibit a high quantum yield of 85% and a narrow full width at half maximum of 16 nm, demonstrating their efficient luminescence in water. Typically, L-Glu on the surface have contributed greatly to an acidic environment and passivation of surface defects, improving the water stability and dispersibility of CsPbBr₃ nanocrystals. Moreover, L-Glu/OAm-CsPbBr₃ NCs exhibit great biocompatibility due to the presence of L-Glu, resulting in their good performance for HeLa cell imaging. Thus, we propose a facile and effective method to prepare CsPbBr₃ nanocrystals with excellent water stability by using L-Glu and OAm as cooperated ligands and expand their application in cell imaging.