Background Dairy cows have a massive demand for glucose at the onset of lactation. A poor adaption to this period leads to an excessive negative energy balance with an increased risk for ketosis and impaired animal health and production. Besides the measurement of ketones, analysing the glucose concentration in blood is reported as helpful instrument for diagnosis and differentiation of ketosis. Monitoring metabolic parameters requires multiple blood sampling. In other species, new blood sampling techniques have been introduced in which small amounts of blood are rapidly analysed using electronic hand-held devices. The objective of this study was to evaluate the suitability of capillary blood for blood glucose measurement in dairy cows using the hand-held devices FreeStyle Precision (FSP, Abbott), GlucoMen LX Plus (GLX, A. Menarini) and the WellionVet GLUCO CALEA, (WGC, MED TRUST). In total, 240 capillary blood samples were obtained from dry and fresh lactating Holstein-Friesian cows. Blood was collected from the skin of the exterior vulva by using a lancet. For method comparison, additional blood samples were taken from a coccygeal vessel and analyzed in a laboratory. Glucose concentrations measured by a standard laboratory method were defined as the criterion standard. Results The Pearson correlation coefficients between the glucose concentrations analyzed in capillary blood with the devices and the reference were 73 % for the FSP, 81 % for the GLX and 41 % for the WGC. Bland- Altman plots showed biases of −18.8 mg/dL for the FSP, -11.2 mg/dL for the GLX and +20.82 mg/dL for the WGC. The optimized threshold determined by a Receiver Operating Characteristics analysis to detect hyperglycemia using the FSP was 43 mg/dL with a sensitivity (Se) and specificity (Sp) of 76 and 80 %. Using the GLX and WGC optimized thresholds were 49 mg/dL (Se = 92 %, Sp = 85 %) and 95 mg/dL (Se = 39 %, Sp = 92 %). Conclusions The results of this study demonstrate good performance characteristics for the GLX and moderate for the FSP to detect hyperglycemia in dairy cows using capillary blood. With the study settings, the WGC was not suitable for determination of glucose concentrations.