The objective of this study was to evaluate the acute response of Sencil™, a fiber-optic sensor, in point accuracy for glucose monitoring in vivo on healthy dogs under anesthesia. A total of four dogs with clinically normal glycemia were implanted with one sensor each in the chest region to measure the interstitial glucose concentration during the ovariohysterectomy procedure. The data was acquired every 10 seconds after initiation, and was compared to the concentration of venous plasma glucose sampled during the surgery procedures for accuracy of agreement analysis. In the four trials with a range of 71–297 mg/dL plasma glucose, the collected 21 pairs of ISF readings from the Sencil™ and the plasma reference showed superior dispersion of residue values than the conventional system, and a linear correlation (the Pearson correlation coefficient is 0.9288 and the y-intercept is 14.22 mg/dL). The MAD (17.6 mg/dL) and RMAD (16.16%) of Sencil™ measurements were in the comparable range of the conventional system. The Clarke error grid analysis indicated that 100% of the paired points were in the clinically acceptable zone A (61.9%) and B (38.1%).