A capillary-based optical biosensor has been developed to detect calpastatin, an indicator of meat tenderness. Longissimus muscle samples (n = 11) were extracted from beef carcasses at 0 and 48 h post-mortem. These samples were assayed for calpastatin by traditional laboratory methods and with a newly developed capillary tube biosensor as well as for Warner-Bratzler shear force (WBSF) and crude protein and the responses were compared. Additionally, the response from the capillary-based biosensor was compared to a previously developed optical fiber biosensor. When the 0 and 48 h sampling periods were combined, the capillary tube biosensor was moderately accurate in predicting calpastatin activity (R2 = 0.6058). There was less variation in the 0 h capillary tube biosensor compared to the 0 h pre-column (P = 0.006) and post-column optical fiber biosensors (P = 0.047), therefore the capillary tube biosensor is a more precise system of measurement. This research further advances the development of a calpastatin biosensor and makes online assessment one step closer to reality.