Background: Phenylalanine hydroxylase activity is deficient in phenylketonuria.  Phenylketonuria is an inherited metabolic disease of protein metabolism. Aim: The study aimed to determine the effect of online education on mothers' knowledge and practice regarding caring for children with phenylketonuria. Design: A quasi-experimental research design was used in this study (pre/post-test). Setting: The research was carried out in a genetic counselling clinic in a basic health care facility affiliated with the Ministry of Health in Sohag City. Sample: A non-probability purposive sampling technique included 50 mothers and their children with Phenylketonuria. Tools: Two tools were used: Tool (I) Self-administering questionnaire included Part (1): Demographic data of the mothers and their children, Part (2): Children's medical history, and Part (3): Mothers' knowledge about phenylketonuria, and Tool (II): Mothers' reported practice about phenylketonuria. Results: The study indicated that there was a statistically significant improvement in mothers' knowledge and reported practices regarding caring for children with phenylketonuria post- online education than pre- online education. A statistically significant correlation was observed between mothers' knowledge and reported practices post- online education. Conclusion: The study results concluded that online education had positive effects on improving mothers' knowledge and practice in caring for their children with phenylketonuria. Recommendations: Online education for mothers regarding caring for children with phenylketonuria is recommended to increase their knowledge and reported practices.