Traditional Chinese medicine (TCM) practitioners assess body constitution (BC) as a treatment basis for maintaining body homeostasis. We investigated patterns in spontaneous brain activity in different BC groups using resting-state functional magnetic resonance imaging (rsfMRI) and determined the relationship between these patterns and quality of life (QOL). Thirty-two healthy individuals divided into two groups (body constitution questionnaire (BCQ)-gentleness [BCQ-G] and BCQ-deficiency [BCQ-D]) based on the body constitution questionnaire (BCQ) underwent rsfMRI to analyze regional homogeneity (ReHo) and the amplitude of low-frequency fluctuation (ALFF). The World Health Organization Quality of Life Instruments (brief edition) scale was used to evaluate the QOL. The BCQ-G group (n = 18) had significantly greater ReHo values in the right postcentral gyrus and lower ALFF values in the brainstem than the BCQ-D group (n = 14). In the BCQ-D group, decreased ReHo of the postcentral gyrus correlated with better physiological functioning; increased ALFF in the brainstem correlated with poor QOL. BCQ-subgroup analysis revealed a nonsignificant correlation between ReHo and Yang deficiency/phlegm and stasis (Phl & STA). Nonetheless, the BCQ-D group showed a positive correlation between ALFF and Phl & STA in the parahippocampus. This study identified differences between BCQ-G and BCQ-D types of healthy adults based on the rsfMRI analysis. The different BCQ types with varied brain endophenotypes may elucidate individualized TCM treatment strategies.