We established and evaluated a polyclonal antibody based competitive enzyme-linked immunosorbent assay for the quantification of defensin-1 in honey. The assay showed an inhibitory concentration (IC₅₀) value of 111.5 ± 15.41 ng/ml with a detection limit of 7.8125 ng/ml. The regaining of defensin-1 in spiked ‘artificial honey’ was between 87.05 and 112.96% with relative standard deviation less than 9.2%. Sensitivity and specificity of the test were experimentally validated on a sample of 20 different honeys. The antibacterial activity of these honey samples showed a significant concentration-dependent correlation with the production of defensin-1 (n = 20; r = −0.6598; P = 0.0016). The assay provides a specific and sensitive method for the screening of defensin-1 in honey. The method to detect honeybee-derived proteins in honey is a promising approach to verifying the authenticity of honey. The defensin-1 ELISA could also be used for the rapid screening of honeys suitable for medicinal purposes.