ABSTRACT Red dragon fruit (Hylocereuspolyrhizus) it is an essential source of betacyanin which is responsible for its red purple pigment. The purpose of this study was to determine the betacyanin content of alcoholic H. polyrhizus (red dragon fruit) flesh extract as an alternative counter stain for semen smear. This study utilized an experimental comparative design method. Samples were subjected with 20ml of 100% methanol in every 100 ml of H. polyrhizus flesh extract and to a series of temperatures: 30oC, 60oC, and 100oC for 5 minutes in the water bath to determine its color stability. This is followed by storing it at low temperature (4oC) without light exposure for 48 hours. The results of this study showed that the pigments were stable at 100oC heat treatment for 5 minutes in water bath and it stained excellently with a rate of 2.48 this signified that the standard eosin Y-nigrosin and alcoholic H. polyrhizus flesh extract have the same color affinity in staining sperm cells. Results also showed that the betacyanin obtained from the H. polyrhizus flesh extract heated at 100oC was effective as an alternative counterstain for semen smear.