Bamboo holds significant potential as a substitute for plastics. Stress relaxation is a critical property during the fixation stage of plastic formation for bamboo products intended to replace plastic. However, research on this topic has been limited. In this study, a custom-built mechanical loading platform coupled with Raman spectroscopy was acquired during stress relaxation of bamboo under different humidity levels. Results reveal variations in stress relaxation patterns based on node position, radial location, and humidity, along with changes in cellulose molecules in response to these factors. Specifically, stress relaxation modulus in bamboo decreases from the outer to the inner part, increases in the presence of node structures, and decreases with increasing humidity. The shifts in Raman spectral bands exhibit significant changes in response to variations in stress and humidity. The elongation of cellulose chain bands at 1097 cm−1 in the Raman spectrum shows an increasing trend during stress relaxation, with larger band shifts observed at lower humidity. Simulations of cellulose molecular stretching show a stronger stress response at lower moisture content, with a maximum stress of 0.85 GPa for cellulose at 3.3 % moisture content, compared to 0.79 GPa at 7.8 % moisture content. These findings suggest a novel approach for the molecular investigation of materials during stress relaxation, which is crucial for the plasticity processing of bamboo.