Background: The anesthetic characteristics of ultrasound-guided bi-level erector spinae plane block (ESPB) plus dexmedetomidine (Dex) remain unclear. We compared the efficacy and safety of ultrasound-guided bi-level ESPB plus different doses of Dex in patients undergoing video-assisted thoracic surgery (VATS). Methods: One-hundred eight patients undergoing VATS were randomized into three groups: R group (n = 38, 15 ml of 0.375% ropivacaine with 0.1 mg/kg dexamethasone), RD1 group (n = 38, 15 ml of 0.375% ropivacaine plus 0.5 μg/kg DEX with 0.1 mg/kg dexamethasone) and RD2 group (n = 38, 15 ml of 0.375% ropivacaine plus 1.0 μg/kg DEX with 0.1 mg/kg dexamethasone). The primary outcome was the pain 12 h after surgery. Secondary outcomes included the Prince Henry Hospital Pain Score; hemodynamics; consumption of sufentanil; anesthetized dermatomal distribution; recovery time; rescue analgesia; satisfaction scores of patients and surgeon; quick recovery index; adverse effects; the prevalence of chronic pain and quality of recovery. Results: The visual analog scale (VAS) and the Prince Henry pain score were significantly lower in both the RD1 and RD2 groups during the first 24 h after surgery (P < 0.05). Both VAS with coughing and the Prince Henry pain score were significantly lower in the RD2 group than in the RD1 group 8-24 h after surgery (P < 0.05). Both heart rate and mean arterial pressure were significantly different from T2 to T6 in the RD1 and RD2 groups (P < 0.05). The receipt of remifentanil, propofol, Dex, and recovery time was significantly reduced in the RD2 group (P < 0.05). The requirement for sufentanil during the 8-72 h after surgery, less rescue medication, and total press times were significantly lower in the RD2 group (P < 0.05). The time to the first dose of rescue ketorolac was significantly longer in the RD2 group (P < 0.05). Further, anal exhaust, removal of chest tubes, and ambulation were significantly shorter in the RD2 group (P < 0.05). The incidence of tachycardia, post-operative nausea and vomiting, and chronic pain was significantly reduced in the RD2 group, while the QoR-40 score was significantly higher in the RD2 group (P < 0.05). Conclusions: Pre-operative bi-level, single-injection ESPB plus 1 μg/kg DEX provided superior pain relief and long-term post-operative recovery for patients undergoing VATS. Clinical Trial Registration: http://www.chictr.org.cn/searchproj.aspx.