Research on the differences in physiological and psychological mechanisms of practitioners in different occupations is a current hot spot, such as pilots. This study explores the frequency-dependent changes of pilots' low-frequency amplitudes in the classical frequency band and sub-frequency band between pilots and general occupations. The goal of the current work is to provide objective brain images for the selection and evaluation of outstanding pilots. Twenty-six pilots and 23 age-, sex-, and education-matched healthy controls were included in this study. Then the mean low-frequency amplitude (mALFF) of the classical frequency band and sub-frequency band was calculated. The two-sample t-test was performed on SPM12 to analyze the differences between the flight group and control group in the classic frequency band. To explore the main effects and the inter-band effects of the mean low-frequency amplitude (mALFF), the mixed design analysis of variance was applied in the sub-frequency bands. Compared with the control group, left cuneiform lobe and the right cerebellum six area of pilots show significant difference in the classic frequency band. And the main effect results in the sub-frequency bands show that the area with higher mALFF in the flight group is located on the left middle occipital gyrus, the left cuneiform lobe, the right superior occipital gyrus, the right superior gyrus, and the left lateral central lobule. However, the area where the value of mALFF decreased is mainly located on the left rectangular cleft with surrounding cortex and the right dorsolateral superior frontal gyrus. Besides, compared with the slow-4 frequency band, the mALFF of the left middle orbital middle frontal gyrus of the slow-5 frequency band was increased, while the mALFF value of the left putamen, left fusiform gyrus, and right thalamus was decreased. The sensitivity of the slow-5 frequency band and the slow-4 frequency band to the pilots' different brain areas was also different. Also, the different brain areas in the classic frequency band and the sub-frequency band were significantly correlated with pilots' flight hours. Our findings showed that the left cuneiform brain area and the right cerebellum of pilots changed significantly during resting state. And there was a positive correlation between the mALFF value of those brain area and flight hours. The comparative analysis of sub-frequency bands found that the slow-5 band could elucidate a wider range of different brain regions, providing new ideas for exploring the brain mechanisms of pilots.