Ultrasound (US) washing has been verified to improve the quality of postharvest blueberry fruit. However, its physiological and molecular mechanisms remain largely unknown. In the present study, an US with a frequency of 25 kHz and a power density of 400 W for 2 min was performed to investigate its role in impacting the quality of blueberries. The results showed that US washing improved the quality of blueberries, with a higher firmness and lignin content (p < 0.05) than the control. Moreover, US washing inhibited the levels of superoxide radical (O2·-) production rate and hydrogen peroxide (H2O2) content while stimulating the superoxide dismutase (SOD) and catalase (CAT) activities of the blueberry fruit. Transcriptomic analysis screened 163 differentially expressed genes (DEGs), and the key DEGs were mainly enriched in phenylpropanoid biosynthesis, flavonoid biosynthesis, and plant-pathogen interaction pathways. Furthermore, the transcription factors and the structural genes associated with lignin biosynthesis were also identified from the DEGs. More importantly, the correlation analysis revealed that firmness and lignin content were positively correlated with the expression of C4H, COMT1, and POD52 in blueberry fruit, indicating that these genes might be involved in the regulation of US-mediated lignin synthesis. The findings provide new insight into the US-enhanced quality of blueberry fruits.