Campylobacter jejuni, a major cause of foodborne zoonotic infections worldwide, shows a paradoxical ability to survive despite its susceptibility to environmental and food-processing stressors. This resilience is likely due to the bacterium entering a viable but non-culturable state, often within biofilms, or even initiating biofilm formation as a survival strategy. This study presents an innovative application of NanoLuc bioluminescence to accurately monitor the development of C. jejuni biofilms on various substrates, such as polystyrene plates, mucin-coated surfaces, and chicken juice matrices. Introduction of NanoLuc luciferase in a pathogenic C. jejuni strain enables rapid non-invasive holistic observation, capturing a spectrum of cell states that may comprise live, damaged, and viable but non-culturable populations. Our comparative analysis with established biofilm quantification methods highlights the specificity, sensitivity, and simplicity of the NanoLuc assay. The assay is efficient and offers precise cell quantification and thus represents an important complementary or alternative method to conventional biofilm monitoring methods. The findings of this study highlight the need for a versatile approach and suggest combining the NanoLuc assay with other methods to gain comprehensive insight into biofilm dynamics. KEY POINTS: • Innovative NanoLuc bioluminescence assay for sophisticated biofilm quantification. • Holistic monitoring of C. jejuni biofilm by capturing live, damaged and VBNC cells. • Potential for improving understanding of biofilm development and structure.