The study was carried out to improve the sensitivity of paper-based biosensors for detecting antibiotics inhibiting bacterial protein synthesis. The biosensor was based on the detection of luminescence intensity of nanoluciferase protein (NanoLuc), which was synthesized on freeze-dried paper containing an in vitro transcription-translation system. NanoLuc was synthesized by applying water samples without antibiotics on freezedried paper, resulting in luminescent emission when combined with the Furimazine substrate. In contrast, in the presence of antibiotics, the luminescent intensity was lower due to the inhibition of NanoLuc synthesis. The luminescent intensity was recorded with a digital camera and quantified by ImageJ software. The estimated detection limits were 13,9; 0,23; 1,2 and 0,32 µg/mL for gentamicin, chloramphenicol, erythromycin, and paromomycin, respectively. The sensitivity of luminescent paper-based biosensors was increased 2 or 3 folds depending on each antibiotic.