Glucose is an analyte of great importance, both in the clinical and sports fields. As an alternative to standard blood analysis, sweat arises as an easily accessible, non-invasive biofluid, as well as a great candidate to monitor human health and sport performance of athletes. However, there is a current need for the fabrication of low cost, easy to use technologies capable of sequential analysis of sweat biomarkers at different times. In this research, we developed a chrono-sampling microfluidic platform integrated with an alginate/TiO2 biosystem for sweat glucose determination. Glucose concentrations ranging from 10 to 1000 µM in artificial sweat were detected and quantified, both in a bead format and in the device, where the whole system was calibrated and verified. The PDMS microfluidic platform allowed chrono-sampling over more than 42 min. This research is oriented towards the development of a miniaturized device for fast analysis at the point and place of need. The integration of the chrono-sampling capacity opens the possibility for a continuous monitoring of sweat biomarkers during exercise, allowing a personalized-oriented training to improve the performance of athletes.