Analysis of the honey enzymes diastase and invertase is one important parameter in honey quality control. Limits for enzyme activity are given by food legislation or directives of national brands, because enzyme activity is one of the measures of adequate conversion of nectar to honey during the ripening process. In addition, certain activity levels of invertase or diastase activity can also act as indicators for heat damage of honey samples. Depending on the botanical origin, enormous differences in enzyme activity can be observed, even though the enzymes are mostly added by the bees. We therefore collected nectar and honey samples during the ripening process of honey and investigated enzyme activity depending on the floral source. Based on the analysis of nectar samples, we could demonstrate that floral source and environmental conditions affect the total sugars and sucrose concentration. We therefore hypothesized that the composition of the nectar, especially the amount of sucrose, may interact with the activity of invertase. We found that correlation between sucrose concentration and invertase activity is highly significant during the ripening process of honey (p < 0.0001) confirming an interaction between these two parameters. This effect was further substantiated by a sugar feeding experiment with defined sucrose concentrations. Possibly a high turn-over at high sucrose concentrations may lead to enzyme exhaustion for invertase.