Methyl violet (CAS number 8004-87-3), also named as Basic Violet, was used as local anti-infective and antiseptic drug as well as sperm staining and counting. This work was formulated to discuss whether methyl violet staining could distinguish between live and death sperm, and in turn, provide a new method for determining sperm survival and service male semen testing and quality evaluation. Over 40 semen samples from healthy male volunteers (aged 20~45) were stained with different concentrations of methyl violet solution (0.01%~0.1%, M/V) and the staining results were observed under optical microscope to distinguish the live sperm and the dead sperm. The amount of total sperm was counted and the survival rate was calculated. The same semen samples were also analyzed with eosin staining (as described by WHO laboratory manual for the examination and processing of human semen) at the same time, and the survival rates from two methods were compared statistically. Results showed that after methyl violet solution staining the survived sperm was mainly light blue under the microscope, while, the dead sperm was mainly purple, and the survival rate calculated was of no significant difference comparing with that of eosin staining method. The results of this study prove that a certain concentration of methyl violet staining can clearly distinguish the live sperm and the dead, further the sperm survival rate of methyl violet was reliable, and can be used as a new analysis of sperm survival evaluation.