Alzheimer's disease (AD) is one of the most challenging diseases around the world with no effective clinical treatment. Previous studies have suggested c-Jun N-terminal kinase 3 (JNK3) as an attractive therapeutic target for AD. Herein, we report 3-substituted indolin-2-one derivatives as the first isoform-selective JNK3 inhibitors by multistage screening. In this study, comparative structure-based virtual screening was performed, and J30-8 was identified with a half-maximal inhibitory concentration of 40 nM, which exhibited over 2500-fold isoform selectivity and marked kinome-wide selectivity. Further study indicated that 1 μM J30-8 exhibited neuroprotective activity in vitro so as to alleviate the spatial memory impairment in vivo through reducing plaque burden and inhibiting the phosphorylation of JNKs, Aβ precursor protein, and Tau protein. All of these indicated J30-8 as proved isoform-selective JNK3 inhibitors that might serve as a useful tool for further JNK3 studies with AD as well as for the development of JNK3 inhibitors for the potential treatment of neurodegenerative diseases.