Abstract The importance of parathyroid glands (PGs) protection is increasingly recognized by thyroid surgeons in the field of minimally invasive video-assisted thyroidectomy (MIVAT). However, current techniques for intra-operative identification and vascularity assessment of the PGs are contentious and complex. This study presents the design and validation of a miniaturized device for fusion near-infrared autofluorescence (NIRAF) based on Raman spectroscopy and laser speckle contrast imaging (LSCI) via an image fusion algorithm for functional protection of PGs in the MIVAT. Our light source components include an integrated light emitting diode (LED) light source fiber, an NIRAF optical fiber, an acquisition optical fiber, and a laser speckle optical fiber in the endoscopic system to achieve identification and vascularity assessment of PGs using a single endoscopic probe. It has been validated in ex vivo tissue experiments that the fluorescence intensity detected by the device was equivalent to that of the marker indocyanine green (ICG) in visual images and superior to that of the thyroid and all other tissues in the neck. Based on clinical studies, MIVAT using functional fluorescence endoscopy, compared with the White light pattern group, the NIRAF combined with LSCI modality group increased the number of intra-operative confirmations of the PGs (P < 0.001), declined the decrease in parathyroid hormone (PTH) (P < 0.05) and calcium levels (P < 0.05) on the first postoperative day, and reduced the incidence of symptomatic hypocalcemia (P < 0.05). Our device may reduce the incidence of postoperative permanent hypoparathyroidism. Application of an miniaturized functional fluorescence endoscope for real-time and label-free PGs identification and vascularity assessment in MIVAT could be realized.