Bacterial infection is a great threat to human health. Lateral flow immunoassays (LFIAs) with the merits of low cost, quick screening, and on-site detection are competitive technologies for bacteria detection, but their detection limits depend on the optical performance of the adopted nanotags. Herein, we presented a LFIA platform for bacteria detection using polydopamine (PDA) functionalized Au nanoparticles (denoted as Au@PDA) as the nanotag. The introduction of PDA could provide enhanced light absorption of Au, as well as numerous functional groups for conjugation. Small recognition molecules i.e. vancomycin (Van) and p-mercaptophenylboronic acid (PMBA) were covalently anchored to Au@PDA, and selected as the specific probes towards Gram-positive (G+) and Gram-negative (G—) bacteria, respectively. Taken Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) as the representative targets of G+ and G— bacteria, two LFA strips were successfully constructed based on the immuno-sandwich principle. They could quantitatively detect S. aureus and E. coli both down to 102 cfu/mL, a very competitive detection limit in comparison with other colorimetric or luminescent probes-based LFIAs. Furthermore, the proposed two strips were applied for the quantitative, accurate, and rapid detection of S. aureus and E. coli in food and human urine samples with good analytical results obtained. In addition, they were integrated as a screening platform for quick evaluation of diverse antibacterial agents within 3 h, which is remarkably shortened compared with that of the two traditional methods i.e. bacterial culture and plate-counting.