[Objective] An analysis of the reason for the yeast flocculation in the process of replicative aging by observation and determination to the physiological properties and cell wall proteins was done in the process of yeast continuous subculture. [Methods] 2-D gel electrophoresis method and infrared spectroscopy method were used to detect the protein on yeast cell wall in the process of continuous subculture. [Results] As the process of subculture continued, the number of sports that could be shown clearly in the 2-D gel electrophoresis picture increased. At the same time, the absorption at the wavelength of 890.51 cm?1 and 808.48 cm?1 decreased in the infrared spectroscopy picture. [Conclusion] In the process of yeast continuous subculture, the degree of glycosylation modification is gradually reducing and the protein groups on the yeast cell wall are getting different which brings changes on the strength of cell wall surface to make the flocculation of yeast stronger.