A new series of C7-substituted chromanones has been designed, synthesized and evaluated for hMAO-B inhibitory activity in vitro. Most of the studied compounds were remarkably potent and selective MAO-B inhibitors, and showed weak or no inhibition of MAO-A. Especially, compound 4f (IC50 = 8.62 nM) was the best MAO-B inhibitor and exhibited the highest selectivity for MAO-B (SI > 11627.9-fold). In addition, the structure-activity relationships for MAO-B inhibition indicated that substitutions at the C7 of the chromanone moiety, particularly with the halogen substituted benzyloxy, were more favorable for MAO-B inhibition. Molecular docking studies have been done to explore the interaction modes of C7-substituted chromanones with MAO-B. Furthermore, the representative compounds 4f and 5d showed low neurotoxicity in SH-SY5Y cells in vitro. So the C7-substituted chromanones could be used to develop promising drug candidates for the therapy of neurodegenerative diseases.