It is an urgent need to develop more effective anti-influenza agents due to the emergence of highly pathogenic and drug-resistant influenza viruses. Herein, a series of 2,4-disubstituted quinazoline derivatives were designed, synthesized and their antiviral activities against influenza A virus were evaluated. Nine compounds (10a2, 16a, 16e, 16i, 16j, 16n, 16o, 16p and 16r) showed potent activity against influenza A virus (IAV) with IC at the low-micromole level (1.29-9.04 μM). Particularly, 16e and 16r possess good anti-IAV activity (IC: 1.29 μM and 3.43 μM, respectively) and acceptable cytotoxicity, and inhibit the transcription and replication of viral RNA. Together with reasonable PK profiles of 16e, these results suggest their promising potential as candidates for further investigation.