In this study, graphene oxide (GO) was prepared for humidity-responsive actuator application. GO has outstanding properties such as single-atom thickness, abundant oxygen, water solubility, and good moisture absorption. GO was prepared at various graphite:potassium permanganate (oxidizer) ratios (1:1, 1:2, 1:3, and 1:4; the corresponding structures are denoted as GO1, GO2, GO3, and GO4, respectively). The GO particles synthesized according to the effect of the oxidizer ratio were characterized to elucidate the internal morphology and humidity response. The defects and disorders and morphological properties of the GO particles were characterized by Raman spectroscopy and transmission electron microscopy (TEM), respectively. To study the humidity-responsive properties, the prepared GO was blended with gelatin (GEL). The bending angle of the GO/GEL hydrogel was studied at relative humidities of 80–85% at 37 ºC. The internal structure morphology of the blend gel was analyzed by synchrotron radiation X-ray tomographic microscopy. GO2/GEL was selected for the development of a humidity-responsive actuator because of its optimal intensity ratio (ID/IG) in the Raman spectrum. X-ray and TEM images showed the good dispersion of GO2 particles in the hydrogel matrix. GO2/GEL exhibited the largest bending angle (θ = 930.11 ± 49.28º) and fast humidity response (angular rate of change ~ 9.380 ± 0.513 ºs-1). Thus, GO2 particles are suitable for use as a humidity-responsive material for humidity actuator applications.