The hamstring muscle group is the most frequently injured muscle group in non-contact muscle injuries in sports involving high-speed running. A total of 84% of hamstring injuries affect the biceps femoris (BF) muscle. Clinical assessments and magnetic resonance imaging (MRI) are routinely used for diagnosis and plan management. MRI-negative scans for clinically diagnosed hamstring injuries range from 14% to 45%. We tested the hypothesis that the functional differences between injured and non-injured BF assessed by tensiomyography can be used for diagnostic and classification purposes. We compared an injured group of 53 international-level soccer players and sprinters with 53 non-injured international-level soccer players and sprinters of both sexes. Comparing the injured vs. non-injured athletes and the left vs. right side in all of the athletes, we used the percentage of absolute differences in the BF contraction time (Tc) to classify non-injured and injured BF muscles. The receiver operating characteristic (ROC) curve and the area under the curve (AUC) and the precision–recall curve (PRC) were used to measure the classification accuracy and to identify cut-off limits using the Tc differences. There was a very high ROC AUC value of 0.981 (SE = 0.009, p < 0.000), with 98.11% of the injured muscles being correctly classified (cut-off point 12.50% on Tc differences), and an AUPRC value of 0.981, with association classification criteria at >9.87. Tensiomyography has a high predictive ability to discriminate between injured and non-injured BF non-invasively and functionally.