In this paper, an accurate U-Pb age of pitchblende was obtained from the No.335 uranium deposit in the Xiazhuang uranium orefield by using the LA-ICPMS technique. The compositional and micro-morphologic characteristics of the pitchblende from the No.335 ore deposit show that it has been altered by some subsequent geological events. (93.5±1.2) Ma is the oldest age in this study. A discussion has been made on the basis of integration of lithosphere extension timing of South China, the control of the diabase dike over the uranium mineralization and the emplacement time of diabase dikes in this ore deposit. It is revealed that the obtained age can reflect some rules of uranium ore formation in South China and might approximately represent the ore-forming age of the No.335 ore deposit.