This study aimed to enhance the bio-ethanol production from date molasses as a cheap and renewable resource by local species of non-Saccharomyces yeasts by optimization the production’s conditions. Hanseniaspora guilliermondii KKUY-0036 and H. uvarum KKUY-0078 were used based on their ability to ferment the date molasses efficiently. They were identified by the sequencing of D1/D2 domain of the 26S rRNA gene. Their identity was confirmed by comparing the obtained sequence with similar sequences allocated in the GenBank. To enhance the ethanol productivity, temperature, pH, fermentation period, molasses concentration and addition of some elements were optimized. Results revealed that the two yeast species exhibited their maximum productivity of ethanol at 30°C when they were grown on 20-25% of the date molasses after 96-120 h of incubation. The highest ethanol concentration was achieved in weak acidic medium (pH 4-6). Addition of zinc, magnesium and manganese induced the production of ethanol by the two yeasts and the optimum concentrations were 0.6, 0.2-0.3 and 0.03 g LG1, respectively. The study introduces both H. guilliermondii KKUY-0036 and H. uvarum KKUY-0078 as new ethanol-producers that ferment date molasses efficiently and will greatly reduce the biofuel production cost.