Lactic acid bacteria (LAB) cause quality deterioration including acidification and production of off-flavor components in food. To rapidly detect LAB, a lateral flow immunochromatographic assay (LFIA) was developed. An antibody (Ab) was obtained using Lactiplantibacillus plantarum as an immunogen, and the LFIA was performed based on this Ab. This showed positive reactions with LAB and a wide range of Gram-positive bacteria. An acid producing ability test using a semiconductor pH sensor was performed in combination with the LFIA. The bacteria testing positive in both the pH test (pH-positive) and the LFIA (LFIA-positive) were LAB. Twenty-three species of LAB in 13 genera could be detected specifically. Fresh foods and their equivalent spoiled foods were cultured for 0, 12, and 24 h, and the time required for the cultures to show LFIA-positive and pH-positive results was investigated. Of the 51 foods, 42 showed shorter culture times for the spoiled food than for the fresh food (spoilage detection rate: 82%). Amplicon analysis detected LAB including Leuconostoc citreum and Leuconostoc gelidum in spoiled foods. As this novel method can detect LAB in approximately 1 h, its use should make it possible to judge the freshness of food before shipping or sale.