In this study, we developed a column-switching high-performance liquid chromatography method with fluorescence detection for the simultaneous analysis of biogenic amines (BAs). This method uses an isocratic solution using acetonitrile with water as the mobile phase. Column-switching is achieved by using a switching valve with a set time program to change flow direction. Using this method, seven BAs (tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, tyramine, and spermidine) could be separated and impurities eliminated. Using fluorescence detection, BAs could be identified with high sensitivity. We employed this method to determine the BA contents of fish (mackerel, tuna, and cod) and fermented food (a soybean product and cheese). Our results are in line with previous reports, yielded highly reproducible and quantitative results, and enabled the quick and simultaneous analysis of multiple BAs. Therefore, the method developed here may be useful for the continuous analysis of BAs in different samples to evaluate food quality.