The aims of this study were to screen and isolate yeast which is producing high biomass and cellulase enzyme from rumen fluid. Two fistulated Thai crossbred Holstein Friesian steers, averaging 350 ± 20 kg body weight, were used. The experiments were designed by 12 × 3 × 3 factorial arrangement in a completely randomized (CRD). Factor A was isolated yeasts (11 difference characteristics) and S. cerevisiae. Factor B was sugarcane molasses (M) concentration (50, 150, and 250 g/L). Factor C was urea (U) concentration (10, 30, and 50 g/L) (experiment 1). To assess biochemical properties, the potential yeast were selected for identified and analyzed. The highest yeast biomass was observed in three yeasts including codes H-KKU20, I-KKU20, and C-KKU20. The highest CMCase activity was observed in yeast code H-KKU20. Identification of isolates H-KKU20 and I-KKU20 revealed that those isolates belonged to Pichia kudriavzevii-KKU20 and Candida tropicalis-KKU20, while C-KKU20 was identified as Galactomyces sp.-KKU20. The P. kudriavzevii-KKU20 and C. tropicalis-KKU20 provided maximum cell growth. The highest ethanol production was observed in S. cerevisiae. The P. kudriavzevii-KKU20 yielded the least reducing sugar. P. kudriavzevii-KKU20 had higher results than the other yeasts in terms of yeast biomass production, cellulase enzyme activity, and cell number. This species may lead to higher production of yeast cell wall than the traditional species in yeast industry (such as S. cerevisiae) or in other applications as ruminant feed additives.