Hemolymph is a liquid of the open circulation system of the honey bee (Apis mellifera L.), which representes a functional equivalent of mammalian blood and lymph. It has a complex chemical composition and contains over 300 different proteins along with sugars and lipid components. Previous studies have revealed certain differences in the hemolymph composition between the honey bee castes, different developmental stages and age groups of bees. However, a detailed chemical characterization of the hemolymph composition has not been performed. Therefore, the aim of this study was to determine the biochemical characteristics and differences in honey bee hemolymph composition depending on the age and gender based on the hemolymph chemical fingerprinting. The study was conducted on 3 experimental honey bee colonies. Hemolymph samples of five age groups of workers (1, 7, 14, 24 and 42 days old; n = 338) and drones (n=8) were collected by thorax extraction from each experimental colony, and analysed by FTIR-ATR spectroscopy. The results showed that honey bee hemolymph provides a characteristic IR spectrum, and based on spectral variations it was determined that the hemolymph of the honey bee does not differ qualitatively depending on age and gender, but there are differences in the proportion of individual compositional components.