The aim of this study was to investigate the effects of ultrasound with different frequency modes on the chemical structure, chain conformation, and immune activity of lentinan from Lentinus edodes; the structure–activity relationship of lentinan was also discussed. The results showed that, compared with original lentinan (extracted using hot water), although ultrasonic treatment did not change the monosaccharide composition and main functional groups of lentinan, it significantly changed its chain conformation. Especially at 60, 40/60, and 20/40/60 kHz, according to atomic force microscopy and solution chain conformation parameters, lentinan transformed from a rigid triple-helix chain to a flexible single-helix chain, and the side-chain was severely broken. Under this condition, lentinan had the worst immune activity. However, at 20/40 and 20/60 kHz, the rigid triple-helix chain transformed into a loose and flexible triple-helix chain, showing the strongest immune activity. These results indicated that dual-frequency ultrasound had significant effects on the conformation of lentinan, and the conformation characteristics of polysaccharide chain such as spiral number, stiffness and flexibility, and side-chain played an important role in immune activity. This study shows the great potential of ultrasound with different frequency modes in carbohydrate food processing, which may have important reference value and practical significance.