European foulbrood (EFB) is a severe bacterial brood disease of honey bees (Apis mellifera) caused by Melissococcus plutonius. Diagnosis of EFB in the field is based on visual inspection of brood-combs and detection of diseased larvae. However, symptoms of EFB may be easily obscured by other diseases or abnormalities in the brood, making definitive diagnosis difficult. Hence, confirmatory laboratory assays, such as PCR and real-time PCR, are used to verify the presence of M plutonius in suspected colonies. While these methods are accurate and specific, they are time consuming and labour intensive. Herein, we report development of a label-free colorimetric nanodiagnostic method for direct detection of unamplified M plutonius DNA using unmodified gold nanoparticles. Under appropriate conditions, the DNA probes hybridised with their complementary target sequences in the sample DNA, which resulted in aggregation of the gold nanoparticles and a concomitant red to blue colour change, which was observed visually. The assay could detect as few as 25 copies of the M plutonius cell wall-associated protease gene within 20 minutes. The assay results were in 100 per cent concordance with real-time PCR-positive and PCR-negative samples. Our study demonstrated that the gold nanoparticles-based assay is a specific and sensitive tool for rapid detection of M plutonius.