Purpose: This observational study assessed sleep disturbance and autonomic dysfunction as risk factors for chronic subjective tinnitus through polysomnography (PSG) and autonomic function tests. Methods: Adult patients with chronic subjective tinnitus who visited the department of otolaryngology in our hospitals (n = 40), along with controls without tinnitus (n = 80), were recruited. Individuals with an average hearing threshold level (HL) exceeding 25 dB HL and a known diagnosis of insomnia were excluded. Objective assessments comprised pure-tone audiometry, PSG, and autonomic function tests (e.g., the cold pressor test). Results: Patients with prolonged sleep latency, lower sleep efficiency, and sympathetic hyperactivity had significantly higher risks of developing tinnitus. No interaction effect between poor sleep quality and sympathetic hyperactivity on tinnitus was detected. Conclusion: This is the first study to administer PSG and autonomic function tests to patients with chronic subjective tinnitus. Poor sleep quality and autonomic dysfunction were implicated as risk factors for tinnitus. PSG and the autonomic function tests helped identify tinnitus-related comorbidities and inform tinnitus treatment. Sleep disturbance and autonomic dysfunction did not exert an interaction effect on tinnitus. Further studies with a larger sample size and the inclusion of patients with more severe tinnitus are warranted.