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Objective This study was performed to evaluate the test–retest reliability of perfusion of the cortex and subcortical white matter on three-dimensional spiral fast spin echo pseudo-continuous arterial spin labeling (3D-ASL). Methods Eight healthy subjects underwent 3D-ASL and structural imaging at the same time each day for 1 week. ASL data acquisition was performed in the resting state and right finger-tapping state. Cerebral blood flow (CBF) images were calculated, and the CBF values of the precentral cortex (PCC) and precentral subcortical white matter (PCSWM) were automatically extracted based on the structural images and CBF images. Results In the resting state, the intraclass correlation coefficient (ICC) of the bilateral PCC was 0.84 (left) and 0.81 (right) and that of the bilateral SCWM was 0.89 (left) and 0.85 (right). In the finger-tapping state, the ICC of the bilateral PCC was 0.91 (left) and 0.87 (right) and that of the bilateral PCSWM was 0.87 (left) and 0.92 (right). The CBF value of the left PCC and PCSWM was not significantly different between the resting state and finger-tapping state on two ASL scans. Conclusion 3D-ASL provides reliable CBF measurement in the cortex and subcortical white matter in the resting or controlled state.