Dye extracts from plants have been valuable not only for the economy but also environmental sustainability. There have been many reports on the utilization of natural dyes extracted from various sources for staining of biological tissues. This study aimed to investigate the extraction of natural dye from black rice (Oryza Sativa), butterfly pea (Clitoria ternatea), fresh roselle (Hibiscus sabdariffa), and mulberry (Morus alba) to stain of human spermatozoa for morphology assessment. The results showed that black rice extracted from solvents C containing 5 ml of absolute ethanol, 10 g of potassium alum and 100 ml of distilled water is the best dye for human spermatozoa evaluation comparable to the rapid PAP and Dip quick® stain. Effectiveness of the process was found with black rice extract stain by using 2 steps for 15 min. There were no statistically significant differences in the parameters of head, midpiece, tail and background for human sperm morphology assessment comparable to rapid PAP and Dip quick® stain (p > 0.05) unless the midpiece compartment when compare to rapid PAP. This finding suggests that the black rice extracted has potential for use as an alternative dye for human spermatozoa morphology evaluation. The usefulness of black rice extracts will decrease the expense for purchasing synthetic dyes and reduce their adverse effects on human and environment.