Objective To investigate the clinical safety and efficacy of ultrasound-guided trioxygen injection of posterior branch of cervical spinal nerve via the lateral approach on the treatment of chronic neck and shoulder pain caused by posterior branch of cervical spinal nerve. Methods A total of 102 patients with chronic neck and shoulder pain caused by posterior branch of cervical spinal nerve admitted to the Department of Pain Medicine, Shanghai Fourth People's Hospital from October 2020 to October 2021 were selected. The patients were randomly divided into group A and B, with 51 cases per group. Using the out-of-plane ultrasound guidance technique, and C3, C4 and C5 posterior branch of spinal nerve as puncture sites, 5 ml of trioxygen (20 μg/ml) was administrated to separate C3, C4 and C5 posterior branch of spinal nerve in patients of group A, and anti-inflammatory and analgesic solutions (betamethasone 1 mg + lidocaine 40 mg, diluted to 12 ml with saline, 2 ml at each point) were injected. Patients in group B were administrated with Xiaoyan Zhentong Liquid into the posterior branch of C3, C4 and C5 spinal nerves alone (the drugs were the same as group A). The pain assessed by the Numerical Rating Scale (NRS), the Pittsburgh Sleep Quality Index (PSQI), head and neck range of motion score (ROM) and complications before treatment and at 1 week, 1 month, 3 months and 6 months after treatment were recorded. Treatment response rate and patient satisfaction with the treatment were recorded as well. Results Compared with those before treatment, there were significant differences in the NRS, PSQI, and ROM scores at 1 week, 1 month, 3 months, and 6 months after treatment between the two groups (P<0.05). There were no significant differences in the NRS, PSQI, and ROM scores at 1 week after treatment between the two groups (P>0.05). There were significant differences in NRS, PSQI, and ROM scores at 1 month, 3 months, and 6 months after treatment between the two groups (P<0.05). Six months after treatment, the patient satisfaction rate was significantly higher in group A than that in group B (75% vs 55%, P<0.05). Local hematoma, infection, regional numbness and other complications immediately after surgery and 3 days after surgery in both groups were not reported. Conclusion Ultrasound-guided trioxygen injection and release of the posterior branch of the cervical spinal nerve via the lateral approach can improve the symptoms of patients with chronic neck and shoulder pain caused by the posterior branch of the cervical spinal nerve for a long time. The puncture approach is safe and the therapeutic effect is exact. It is an effective treatment.