To verify the effect of sleep ambient music intervention (SAMI) on sleep quality and mental status of college students, and to further explore the minimum effective duration of SAMI, this study was designed as a pre-and post-intervention self-controlled exploratory study. Participants were subjected to a one-week no-intervention test, then 4 weeks of music intervention followed. Subjective sleep quality data were collected using the Pittsburgh Sleep Quality Index (PSQI); objective sleep quality data were collected with Actigraphy; and mental status data were collected using the State–Trait Anxiety Inventory (STAI) and the Beck Depression Inventory-II (BDI-II). Data were analyzed and processed using mixed-effects models and repeated measures. The results showed that compared with the no-intervention week, college students’ subjective sleep quality, objective sleep onset latency (SOL), trait anxiety, and depression symptom were reduced at week 1; week 2; week 3; week 4 under SAMI; state anxiety of college students at week 3 and week 4 under SAMI were also reduced. And there were differences in sleep quality among college students of different genders too. Compared with females, males had worse sleep efficiency (SE), shorter total sleep time (TST), and more awaking times (AT). In addition, 3 days was the minimum effective length for SAMI to shorten objective SOL, and 2 days was the minimum effective length to shorten the subjective SOL of college students. The findings of this study suggest that SAMI can improve subjective sleep quality, shorten objective SOL, and reduce anxiety and depression in college students. Interventions for more than 3 days had a significant effect on shortening SOL and long-term effects seemed to emerge after 3 weeks.