Microfluidic devices that generate stable concentration gradients are efficient instruments for automated calibration for analytical and bioanalytical systems. However, little attention has been paid to the development of reusable microfluidic concentration gradient generators, which can be useful for a range of species through mathematical characterization. In this work, we develop a microfluidic device based on three steps of serial dilution that were able to generate nonlinear concentration gradient for dyes and biomolecules. The microfluidic device was described mathematically, statistically and was suitable for reusable analytical and bioanalytical analysis. The device reproducibility was assessed by experimental tests, which have shown the same gradient concentration profile for different dyes and statistical reproducibility with 95% confidence interval for bovine serum albumin (BSA). Moreover, the experimental data converged well with those obtained by computational fluid dynamics simulation. Applicability was verified by coupling the microfluidic device to a surface plasmon resonance (SPR) biosensor, based on nanohole arrays with sensitivity of 358.7 nm RIU −1 determined by white-light SPR excitation exposed to different D-(+)-glucose aqueous solutions with 1.3361-1.4035 refractive index interval. The transmission light intensities obtained by the array of images allowed to quantify a pseudo-unknown BSA sample (160 µg mL −1 ) at 138 µg mL −1 . The SPR analysis has been validated in parallel by fluorescence emissions, which showed a concentration of 154.8 ± 16.6 µg mL −1 .