Background: The objective and accurate assessment of children’s sedentary and physical behavior is important for investigating their relation to health. The purpose of this study is to validate a simple and robust method for the identification of sitting, standing, walking, running and biking performed by preschool children, children and adolescents in the age from 3 to 16 years from a single thigh-worn accelerometer. Method: A total of 96 children were included in the study and all subjects followed a structured activity protocol performed in the subject’s normal kindergarten or school environment. Thigh acceleration was measured using the Axivity AX3 (Axivity, Newcastle, UK) device. Method development and accuracy was evaluated by equally dividing the subjects into a development and test group. Results: The sensitivity and specificity for identifying sitting and standing was above 99.3% and for walking and running above 82.6% for all age groups. The sensitivity and specificity for identifying biking was above 85.8% for children and adolescents and above 64.8% for the preschool group using running bikes. Conclusion: The accurate assessment of sitting, standing, walking, running and biking from thigh acceleration and with children in the age range of 3 to 16 is valid, although not with preschool children using running bikes.