Abstract: The Shijuligou deposit was separated by an arcuate ductile shear zone cross the center of the deposit region, resulting in the difference between the southern and northern ore bodies. The lead (Pb) isotopic data of ores of the Shijuligou copper deposit have averages of 206Pb/204Pb, 207Pb/204Pb, and 208Pb/204Pb in 17.634, 15.444, and 37.312, respectively. It has been shown that ore-forming metals originated from intrusive and extrusive rocks in the upper part of ophiolites. The sulfur isotopic data of pyrite and chalcopyrite in the northern part change from +7.61‰ to +8.09‰ and +4.95‰ to +8.88‰ in the southern part. Isotopes of δ18O in the Shijuligou copper deposit are between +11.1‰ and +18.6‰, with the calculated δ18OH2O at +0.65‰. It is suggested that the mineralized fluid is a mixture of magma fluid, meteorological water, and seawater through circulating and leaching metals from the volcanic rocks. The zircon uranium-lead (U–Pb) dating of gabbro is 457.9±1.2 Ma, and the lower crossing age of the discordant and concordia curves of pyroxene spilite of zircon is 454±15 Ma. It is indicated that the Shijuligou deposit formed in a new ocean crust (ophiolite) of the back-arc basin in the late Ordovician. Mineralization should occur in the intermittence period after strong volcanic activity, and the age should be the late Ordovician. Moreover, the mineralization of ophiolite-hosted massive sulfide deposits in the ancient orogenic belt of the late Ordovician in the northern Qilian Mountains was controlled by the primary fault/fracture, with the forming of a metallogenic hydrothermal system by a mixture of volcanic magma fluid and seawater, which circularly leached the metallogenic metals from the volcanic rocks, resulting in their accumulation. The ore bodies were transformed with morphology and metallogenic elements. Jasperoid is an important sign for prospecting such deposits. There were many island arcs in the continent of China. This study provides evidence for understanding and exploration of ophiolite-hosted massive sulfide deposits in western China, especially in the area of northern Qilian Mountains.