A series of 4-thiazolinone derivatives (D1-D58) were designed and synthesized. All of the derivatives were evaluated in vitro for neuraminidase (NA) inhibitory activities against influenza virus A (H1N1), and the inhibitory activities of the five most potent compounds were further evaluated on NA from two different influenza viral subtypes (H3N2 and B), and then their in vitro anti-viral activities were evaluated using the cytopathic effect (CPE) reduction assay. The results showed that the majority of the target compounds exhibited moderate to good NA inhibitory activity. Compound D18 presented the most potent inhibitory activity with IC values of 13.06 μM against influenza H1N1 subtype. Among the selected compounds, D18 and D41 turned out to be the most potent inhibitors against influenza virus H3N2 subtype (IC = 15.00 μM and IC = 14.97 μM, respectively). D25 was the most potent compound against influenza B subtype (IC = 16.09 μM). In addition, D41 showed low toxicity and greater potency than reference compounds Oseltamivir and Amantadine against N1-H275Y variant in cellular assays. The structure-activity relationship (SAR) analysis showed that introducing 4-COH, 4-OH, 3-OCH-4-OH substituted benzyl methylene can greatly improve the activity of 4-thiazolinones. Further SAR analysis indicated that 4-thiazolinone and ferulic acid fragments are necessary fragments of target compounds for inhibiting NA. Molecular docking was performed to study the interaction between compound D41 and the active site of NA. This study may providing important information for new drug development for anti-influenza virus including mutant influenza virus.