Background: Changes in glycemic levels can negatively affect the body. Several techniques for the measurement of blood glucose have been described, but the enzymatic method is considered standard and more accurate in both humans and animals. The College of American Pathologists recommends the use of portable glucometers (PGs), which are routinely used in human medicine because this is an easy, relatively inexpensive method that delivers results quickly. The aim of this study was to compare the results of the measurement of blood glucose in cattle obtained using portable Accu-Check® glucometer with the enzymatic method (EM), which is still considered standard.Materials, Methods & Results: Thirty crossbred cattle (Bos taurus), male and female, of different ages were evaluated. Through a puncture of the jugular vein, 10 mL of blood was collected, and one drop was immediately used in an Accu-Chek® PG; the remaining blood was stored in tubes containing sodium fluoride and tubes containing EDTA. The samples were packaged and sent to the laboratory for processing. Blood glucose was measured in the sodium fluoride samples using the enzymatic-colorimetric method (EM) employing Labtest® glucose kits with automatic ELISA spectrophotometer readings. The glycemic values obtained in this study with PG and EM ranged from 62 to 163.3 mg/dL. Mean glucose concentrations for the PG and EM methods were 93.53 mg/dL and 94.84 mg/dL, respectively, with no statistical difference (P > 0.05). The glycemia measurement results generated by both tests were normally distributed by the Shapiro–Wilk test (P > 0.05) and equal variances by the Levene test (P > 0.05). Discussion: The glycemic values obtained in this study meant that most of the mean glycemic values evaluated were >45 to 75 mg/dL, considered a reference for the species. This may have occurred due to the stress of handling during sample collection. Some authors compared the GP and EM methods and reported that the mean glucose concentrations obtained using PGs were significantly lower than those using the EM in both cattle and sheep and suggested using a correction factor for PGs. The error rates between PG and EM in our study ranged from 1.7 to 7.8%, much lower than the limits set by the Food and Drug Administration, which stipulates that PG cannot have error rates greater than 20% for blood glucose concentration. Comparison of PG and EM efficacy has been reported for dogs, cats, and horses, and significant differences were observed in the statistical analysis. In studies from other authors concluded that PG provides significantly different results from EM in the measurement of bovine and sheep blood glucose, but the final values can be corrected in order to obtain reliable results to be used in clinical practice. Hematocrit below 30% leads to erroneously high results, whereas hematocrit greater than 55% may give erroneously low results. However, failures are mainly caused by the user, such as improper application of the sample, excessive time to perform the exam, lack of equipment maintenance, and improper storage of test strips. PGs are becoming a very useful tool in the clinical practice with small animals, as studies have shown its reliability in relation to the EM, which is considered the gold standard. This practice may also be extended to cattle due to the reliability of PGs, as indicated in this study.