We improved our previous micro-device containing motile micro-algae (Euglena gracilis) cells for toxicity monitoring, by introducing micro-trenches between a micro-aquarium and two surrounding channels. Target test solution was introduced from the bypass channel to the micro-aquarium through micro-trenches, with a pulse injection and continuous diffusion. Although the chemical gradient formed in the micro-aquarium was not permanently sustainable but only temporal for a couple of 10 min, the device was simple, easy-to-use, and cost-effective to evaluate the toxicity of the test solution. The cells showed their chemotaxis to the temporal chemical gradients, resulting in the deviated distribution of the cells in the micro-aquarium, whereas metabolic disturbing effects of toxic substances induced the unusual motion of the cells.