Salmonella enterica is an important foodborne pathogen. Its rapid and reliable detection in food is critical for the effective prevention of the outbreak of foodborne diseases. Here, we report a novel method for the rapid, specific, label‐free, and real‐time detection of S. enterica with biolayer interferometry (BLI) using an antibody as the receptor. The limit of detection for S. enterica in buffer was 1.6 × 105 CFU/ml at a binding time of 300 s. This method enabled the successful identification of S. enterica in complex food matrices (whole egg powder and beef samples) with satisfactory recovery rates. This BLI‐based method holds great promise in serving as a novel diagnostic tool in the field of food safety and other related fields for the rapid, label‐free, specific, and real‐time detection of S. enterica.