The Wuhe area in eastern Anhui province, hosting 23 metric tons of gold, has been recognized as a new gold ore cluster in the North China Craton (NCC) since the Hekou gold deposit was discovered in 2017. Its metallogenic setting and ore-forming process remain unclear. In this contribution, we perform a combined geology, cathodoluminescence, fluid inclusion, H-O isotope and pyrite Rb-Sr geochronologic studies on the Hekou gold deposit of the Wuhe area. In order to perform detailed fluid inclusion petrography study, we used scanning electron microscope-cathodoluminescence (SEM-CL) on a well preserved sulfides-bearing quartz grain from the auriferous quartz vein. In this quartz grain, generation A barren quartz in the core zone (QA, CL-white), minor sulfides bearing generation B (QB, CL-light gray) in intermediate zone and sulfides rich generation C (QC, CL-dark gray) in the distal zone and fractures were revealed. Only in QC, the higher carbonic proportion (50–80 vol%) inclusions (CC) coexist with the two-phase liquid-rich aqueous fluid inclusions (LC) and sulfides, meanwhile the CC and the LC show a similar Th of 258–313 °C and 249–308 °C, but different salinities of 1.4–5.3 wt% NaCl equivalent and 6.0–10.9 wt% NaCl equivalent, respectively. It suggests that dominating fluid immiscibility recorded in QC is responsible for the prime deposition of gold and sulfide minerals. The δ18O (-4.1 to −6.5‰) and δD (-67.1 to −81.3‰) values of fluid inclusions in quartz suggest that ore-forming fluids were mainly derived from magmatic water which could related to mantle source. Gold-bearing pyrites yield an Rb-Sr isochron age of 128.1 ± 2.7 Ma, which is 8 m.y. earlier than the peak value of gold mineralization ages (ca. 120 Ma) at Jiaodong in response to the Early Cretaceous lithospheric thinning of the NCC and the reactivation of the Tan-Lu Fault zone (TLFZ). The Wuhe area shares similar characteristics of geology, fluid inclusions, H-O isotopes, and ore-forming ages with the Jiaodong gold ore cluster, highlighting the exploration potential in this region.