Low-cost mini-bioreactor platforms are becoming increasingly important in synthetic biology and biotechnology for the characterization of genetic constructs and their initial scaling-up to large-scale cultures. Key process variables are the specific growth rate of cells and the synthesis rate of reporter proteins associated with transcriptional units of interest. These variables are of paramount importance for the characterization of gene synthetic circuits. In addition, their on-line estimation can be used for real-time monitoring of cells’ metabolic state and gene circuit dynamic performance, thus allowing for on-line decision taking. In this work, we first describe a procedure for the calibration of absorbance and fluorescence measurements, ensuring standardized and comparable units across different experimental setups and measurement devices. Then, we implement an observer-based software sensor that uses the calibrated on-line measurements to simultaneously estimate the specific growth and protein synthesis rates. We implemented the calibration procedure and software sensor in a Chi.Bio mini-bioreactor platform. The experimental results show very good performance and pave the way for the use of mini-bioreactor platforms in the real-time characterization of gene synthetic circuits under dynamically regulated time-varying complex scenarios.