Abstract: Daphnia magna is a key model organism in aquatic toxicology and water quality assessment. Behavioral responses in D. magna provide a more rapid and sensitive indicator of toxicant exposure compared to conventional endpoints, making them valuable for Real-time biomonitoring. However, the lack of standardized testing conditions-particularly concerning the spatial dimensions of exposure vessels-undermines the reliability and comparability of toxicity evaluations. In this study, multi-well plates with varying well diameters (6, 10.5, 15, 20, 28, and 37.5 mm) were used to systematically examine how spatial confinement influences locomotor parameters (average velocity, average acceleration, and fractal dimension) in D. magna, both under non-toxic conditions and upon exposure to trichlorfon (TCF). Additional validation tests were conducted using copper sulfate (CuSO₄) and fluoxetine (FLX). The study aimed to clarify how movement space affects baseline behavior in non-toxic settings and modulates toxic responses under chemical stress, ultimately identifying optimal conditions that balance sensitivity, stability, and throughput. Results indicated that under non-toxic conditions, the 20 mm diameter wells produced the most stable baseline locomotion, with minimal rates of changes in average velocity and fractal dimension over 35 minutes (3.98% and 0.63%, respectively). Moreover, the relative standard deviation of average velocity (RSD v̄ =30.1%) was significantly reduced (by 32.5%) compared to that observed in 6 mm wells. When exposure to 6.8 μg/L TCF, 20 mm well elicited the strongest toxic response, with an average velocity inhibition rate of 41.3% and the highest response efficacy index (REI=1.65). Validation experiments with CuSO₄ and FLX further confirmed that the 20 mm condition induced the greatest response magnitude (rates of change is –38.6% and –29.3%, respectively) and the lowest RSD values (28.0% and 24.3%, respectively). Therefore, a well diameter of 20 mm is identified as a critical threshold that minimizes spatial constraints while optimizing detection sensitivity and data stability. This study provides essential theoretical and experimental support for standardizing behavioral bioassays in D. magna, particularly in defining appropriate spatial parameters.