A micro-sequential injection spectrophotometric procedure for DNA assay was developed based on the employment of a lab-on-valve (LOV) meso-fluidic analytical system. A small amount of crystal violet solution (10μl) was de-colored inside the flow cell of the LOV at the presence of 5μl λ-DNA/HindIII within a certain pH range, and the absorbance decrease of crystal violet solution at 591 nm was measured via optical fibers and was employed as the basis of quantification. A uni-variant approach was adopted for the optimization of experimental parameters, including buffer pH, concentration and volume of crystal violet solution, reaction time and sample/reagent loading flow rates. A linear calibration graph was obtained within 0.2-6.0μgml−1, along with a detection limit of 0.07μgml−1. The procedure was applied for the determination of λ-DNA/HindIII in synthetic samples in comparison with a documented procedure.