Ophiolite-hosted diamonds have been discovered in several ophiolites within the Neo-Tethyan tectonic belt, however, the presence of diamonds in Paleo-Tethyan ophiolites remains unclear. This study focuses on the 550~495 Ma Yushigou ophiolite within the North Qilian orogenic belt. Heavy mineral separation and petrological studies of mantle peridotites were conducted. The results reveal the separation of 25 diamond-grains and other unusual minerals from approximately 510 kg of harzburgite. The diamonds mainly range in size from 50 to 150 μm, exhibiting colorless to pale yellow hues, and display crystal forms including octahedral, broken irregular, and rounded shapes. Raman spectroscopy shows a characteristic peak at 1330.37 cm?1. The host harzburgite consists mainly of olivine (75%~85%), orthopyroxene (14%~24%), and minor chromian spinel (<1%). Olivine has Fo values of 90.9~93.23, classifying it as forsterite, with the highest Fo value (93.23) observed in olivine inclusions within chromian spinel. Orthopyroxene occurs primarily in two types of porphyroclastic and intergranular. Porphyroclastic orthopyroxene has Mg# values of 91.07~92.59, Al?O? contents of 0.94~1.92%, and Cr?O? contents of 0.43~0.73%. Intergranular orthopyroxene has Mg# values of 91.40~92.31, Al?O? contents of 1.09%~1.80%, and Cr?O? contents of 0.46%~0.65%. The variation ranges of Mg#, Al?O?, and Cr?O? in porphyroclastic orthopyroxene are all slightly larger than those in intergranular orthopyroxene. Chromian spinel has Cr# values of 56.14~70.21, Mg# values of 43.89~58.33, and TiO? contents below 0.05%. The Mg#-Cr# diagram of spinel indicates that the harzburgite underwent a high degree of partial melting (25%~35%) and experienced metasomatism by melts/fluids in a forearc subduction zone. Ultrahigh-pressure minerals like diamonds suggest formation depths >150 km. This implies that oceanic crust/mantle materials were transported into the deep mantle during subduction. Diamonds and other minerals formed under high-pressure and highly reducing conditions, were encapsulated by mantle peridotite and chromitite, and then were subsequently carried to the surface during the exhumation process. This study confirms the globally significant distribution of ophiolite-hosted diamonds and provides new insights into deep subduction processes and deep mantle material recycling within the North Qilian orogenic belt.