Intolerance of Uncertainty (IU) is a transdiagnostic factor involved in several psychological disorders. Adolescence is characterized by elevated uncertainty and psychopathological vulnerability, but insufficient attention has been paid to IU at this age. This study aimed to investigate the factor structure and psychometric properties of the Intolerance of Uncertainty Scale-Revised (IUS-R) in Italian preadolescents and adolescents. 862 Italian students (57.3% girls) aged 11–17 (M = 14.8 ± 1.91) completed the IUS-R and measures of internalizing and externalizing symptoms, and psychological well-being. To test the factor structure of the IUS-R, one-factor, two-factor, and bifactor models were compared; measurement invariance, reliability, and validity were also addressed. Results showed that the bifactor model outperformed alternative factor models, and a general factor was needed to model the IUS-R. Bifactor model indices supported using the total score to assess IU reliably. Configural and metric invariance by age and sex were fully supported, while the IUS-R achieved partial scalar invariance. Significant correlations emerged for the IUS-R total score with psychopathological constructs, while no relationships with psychological well-being were found. Compared to adult normative data, higher total IUS-R scores were detected, suggesting that IU may be a phase-specific characteristic of adolescence. Our findings support using the IUS-R to measure IU across the lifespan. The recommended use of the total score and its associations with psychopathological dimensions highlight the transdiagnostic nature of IU in adolescence. Therefore, including IU when implementing interventions to prevent maladaptive outcomes in teenagers would be beneficial.