This study describes the development of acetylcholinesterase (AChE), carboxylesterases (CaE1, CaE2, CaE3), glutathion-S-transferase (GST), alkaline phosphatase (ALP) and catalase (CAT) as enzyme biomarkers of exposure to xenobiotics such as thiamethoxam in the honey bee Apis mellifera. Extraction efficiency, stability under freezing and biological variability were studied. The extraction procedure achieved good recovery rates in one extraction step and ranged from 65 percent (AChE) to 97.3 percent (GST). Most of the enzymes were stable at −20 °C, except ALP that displayed a slight but progressive decrease in its activity. Modifications of enzyme activities were considered after exposure to thiamethoxam at the lethal dose 50 percent (LD50, 51.16 ng bee−1) and two sublethal doses, LD50/10 (5.12 ng bee−1) and LD50/20 (2.56 ng bee−1). The biomarker responses revealed that, even at the lowest dose used, exposure to thiamethoxam elicited sublethal effects and modified the activity of CaEs, GST, CAT and ALP. Different patterns of biomarker responses were observed: no response for AChE, an increase for GST and CAT, and differential effects for CaEs isoforms with a decrease in CaE1 and CaE3 and an increase in CaE2. ALP and CaE3 displayed contrasting variations but only at 2.56 ng bee−1. We consider that this profile of biomarker variation could represent a useful fingerprint to characterise exposure to thiamethoxam in the honey bee A. mellifera. This battery of honey bee biomarkers might be a promising option to biomonitor the health of aerial and terrestrial ecosystems and to generate valuable information on the modes of action of pesticides.