The low sensitivity of Lateral flow assay (LFA) limits its application in rapid detection for trace targets. LFAs with nanozyme (nanozyme-LFA) as signal labels have demonstrated excellent performance in point of care testing (POCT). However, additional operational steps for substrate catalysis in nanozyme LFA are required, which makes the nanozyme-LFA operation complicated. In this work, we designed a LFA based on delayed substrate release (SGF-LFA), in which a commercialized glass fiber membrane embedded with substrate (SGF) was fixed at the sample pad. The SGF could automatically execute substrate delivery and catalysis, thus eventually achieving a one-step LFA operation for the nucleic acid detection of influenza A virus H1N1. In this SGF-LFA, 3,3 '- diaminobenzidine (DAB) was oxidized and deposited, producing a strong signal amplification under the catalysis of Au@PtNP nanozyme. The SGF-LFA could detect the nucleic acid of H1N1, with a linear range of 0.02-50 nM and a limit of detection (LOD) as low as 0.02 nM, which was 25-fold lower than that of the nanozyme-LFA before catalysis. In addition, the analytical performance was close to that of a manual operation mode of catalysis amplification. The application of SGF-LFA for detecting the H1N1 nucleic acid in serum samples obtained a recovery rate of 96 %-102.7 %, indicating that SGF-LFA has great potential in point-of-care testing.