In order to reduce the large calibration matrix usually required for calibrating multiwavelength optical sensors, a simple algorithm based on the addition in process of new standards is proposed. A small calibration model, based on 14 standards, is periodically updated by spectra collected on-line during fermentation operation. Concentrations related to these spectra are reconciled into best-estimated values, by considering carbon and oxygen balances. Using this method, fructose, acetate, and gluconacetan were monitored during batch fermentations of Gluconacetobacter xylinus 12281 using mid-infrared spectroscopy. It is shown that this algorithm compensates for noncalibrated events such as production or consumption of by-products. The standard error of prediction (SEP) values were 0.99, 0.10, and 0.90 g/L for fructose, acetate, and gluconacetan, respectively. By contrast, without an updating of the calibration model, the SEP values were 2.46, 0.92, and 1.04 g/L for fructose, acetate, and gluconacetan, respectively. Using only 14 standards, it was therefore possible to approach the performance of an 88-standard-based calibration model having SEP values of 1.11, 0.37, and 0.79 g/L for fructose, acetate, and gluconacetan, respectively. Therefore, the proposed algorithm is a valuable approach to reduce the calibration time of multiwavelength optical sensors.