Objective To evaluate the intraoperative identification of ectopic parathyroid tissue in the central neck region using autofluorescence point-spectral analysis （AFPSA） combined with immune colloidal gold technique （ICGT）， for guiding total parathyroidectomy （TPTX） or clean parathyroidectomy （CPTX） in the management of secondary hyperparathyroidism （SHPT）. Methods Retrospectively collected and compared the clinical data of 64 patients with SHPT from October 2019 to June 2023. In the observation group， TPTX was performed as the initial procedure in 36 cases， followed by sampling of suspicious targets using AFPSA in the central neck area and subsequent detection through ICGT. CPTX was then conducted if a positive result was obtained. On the other hand， the control group consisted of 28 cases where only TPTX was performed without any additional tests during surgery. The surgical data， parathyroid hormone （PTH） levels， blood calcium levels， blood phosphorus levels， alkaline phosphatase （ALP） levels， regression of clinical symptoms， changes in parathyroid function and occurrence of hypocalcemia were compared between these two groups. Results In the observation group， there were 9 cases of AFPSA-ICGT positivity， including 2 left-sided cases， 4 right-sided cases， and 3 thymic cases； among these positive cases， there were a total of 10 locations with mildly hyperplastic or nonhyperplastic microscopic parathyroid tissue. The difference in the number of total parathyroid glands removed （including ectopic） between the two groups was statistically significant （P < 0.05）. At both 3 and 6 months postoperatively， ALP levels in the observation group were significantly lower than those in the control group （P < 0.01 and P < 0.001 respectively）； at 6 months postoperatively， differences in PTH and blood phosphorus levels between the two groups were also statistically significant （P < 0.05 and P < 0.001 respectively）. Joint bone pain and skin itching recurred in some patients within the control group at six months after surgery （P < 0.05）， whereas recurrence of SHPT was less frequent within the observation group compared to controls （P < 0.05）； however， no statistically significant differences were observed regarding postoperative hypoparathyroidism or hyperparathyroidism as well as hypocalcemia between either groups. Conclusion The AFPSA-ICGT intraoperative test can be utilized to guide surgery for SHPT， enabling accurate and efficient identification as well as safe targeting of parathyroid tissues that may not exhibit obvious hyperplasia in the central cervical region.