Oxidative stress leads to an increase in the production of reactive oxygen species in cells and can be induced by environmental factors. To study free radical production in living microalgae, we use time-resolved microfluorimetry, a technology adopted from research on mammalian cells. In contrast to fluorescent probe-based measurements that rely on intensity changes, our sensor detects the presence of free radicals through collisional quenching, and is insensitive to most artifacts commonly observed with intensity-based methods. A new probe, 1-pyrenebutanol allows estimation of free radicals production in the green microalga Tetraselmis ssp., for the first time. In addition, our method monitors simultaneously metabolic rate (through bound-free NAD(P)H ratio). Our results show that free radical production in algal cells is correlated to algal aging, and that during cell growth phases both intracellular free radicals and metabolic activity increase. Concerning thermal stress, we observe that rapid and low temperature changes (<10°C) induce instantaneously an increase in ROS production. Our findings provide new insights into the production of free radicals in response to environmental stresses in unicellular green algae.