Abstract: As space biology experiments transition from short-term post-flight observations to long-term in-orbit monitoring, particularly with the application of the extravehicular radiation exposure platform on the Chinese Space Station, understanding the long-term biological effects of space radiation has emerged as an urgent research direction in space life sciences. Caenorhabditis elegans (C. elegans), which shares 60%～80% of homologous genes with humans, serves as a model organism for studying radiation effects in spaceflight experiments, providing essential insights for assessing and mitigating radiation risks in deep-space exploration. To enable long-term analysis of C. elegans development both inside and outside the spacecraft, a microfluidic chip-based liquid culture system can be utilized for single-individual worm loading and observation. Microfluidic chips regulate the number of nematodes entering the culture chamber by controlling the inner diameter of the channels. Therefore, the preparation and loading of chip samples impose precise requirements on the body width of nematodes, which is directly related to their developmental stage. To clarify the developmental requirements and loading protocols for nematode samples in microfluidic chips, this study measured the body width of wild-type (N2), DNA damage repair proteins (RAD-51, CEP-1), and muscle motility proteins (UNC-54) fluorescent strains of nematodes during different population proliferation cycles and larval development using the heat-shock method. This approach determined the optimal proliferation duration and developmental period for different nematode strains, addressing the issue of insufficient sample synchronization during microfluidic chip loading. After preparation using the aforementioned protocol, four types of nematodes were loaded onto the nematode chip for the Shenzhou 16 mission. The body width of the samples ranged from 27.71 μm to 28.02 μm, which meets the chip loading requirement of 24 μm to 29 μm and ensures consistency in the individual states within the chip. This finding validates the feasibility of the preparation protocol. This study established an experimental workflow for nematode “proliferation-synchronization-body width control-chip loading”, and proposed “quality control” requirements for nematode cultivation and observation in liquid culture systems aboard space stations.