Neurodevelopmental disorders greatly impede the growth and development of infants and young children. Therefore, active substances that can improve locomotion and neurodevelopment in infants and young children are urgently needed. The effects of 6′-sialyllactose (6′-SL), known for its role in improving intestinal health, were investigated in relation to neurodevelopment and locomotion ability in Caenorhabditis elegans. The results showed that treatment with 0.02~1.00 mg/mL 6′-SL extended the paralysis lifespan of C. elegans under high-temperature conditions. At the optimal concentration of 6′-SL, the average, median, and maximum lifespans of C. elegans were 8.70, 8.67, and 12.67 h, respectively. Based on practical application experience, a concentration of 0.56 mg/mL was selected for further research. The analysis showed that 0.56 mg/mL 6'-SL significantly increased the head swing frequency and pharyngeal pumping rate of CL4176 by 7.39% and 4.25%, respectively, and increased the locomotion rate by 16.27%. 6′-SL significantly protected the morphology of cholinergic neurons in C. elegans. In addition, 6′-SL reduced acetylcholinesterase activity by 22.60% and increased dopamine, serotonin, 4-aminobutyric acid, and glutamate levels by 148.71%, 72.17%, 34.72%, and 68.47%, respectively. In summary, 6′-SL can improve locomotion ability by increasing the head swing frequency, pharyngeal pumping rate, and movement rate. 6′-SL also promotes neurodevelopment by inhibiting acetylcholinesterase activity and increasing the neurotransmitter content in C. elegans. These findings provide theoretical support for the application of 6′-SL in formula and health products for infants and young children.