To enhance food safety, the need for swift and precise detection of B. licheniformis, a bacterium prevalent in various environments, including soil and food products, is paramount. This study presents an innovative and cost-effective bioassay designed to specifically identify the foodborne pathogen, B. licheniformis, utilizing a colorimetric signal approach. The biosensor, featuring a 3D-printed architecture, incorporates a casein-based liquid-proof gelatine film, selectively liquefying in response to the caseinolytic/proteolytic activity of external enzymes from the pathogen. As the sample liquefies, it progresses through a color layer, causing the migration of dye to an absorbent layer, resulting in a distinct positive signal. This bioassay exhibits exceptional sensitivity, detecting concentrations as low as 1 CFU/mL within a 9.3-h assay duration. Notably, this cost-efficient bioassay outperforms conventional methods in terms of efficacy and cost-effectiveness, offering a straightforward solution for promptly detecting B. licheniformis in food samples. Display omitted •A cost-efficient and highly specific bioassay tailored for detecting the foodborne pathogen, B. licheniformis•Single-step analysis the caseinolytic/proteolytic enzymatic reactions exhibited by B. licheniformis on the casein/gelatin layer•A simple, budget-friendly, and portable biosensor to detect Bacillus licheniformis•The biosensor may specifically detect Bacillus licheniformis in different foods