Autism spectrum disorder (ASD) is a neurodevelopmental disorder in which early diagnosis is critical for effective intervention and improved outcomes. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and have emerged as promising biomarkers for neurological disorders, including ASD. In our previous discovery study, we identified dysregulated expression of several miRNAs in the plasma samples of children with ASD aged 5-12 years. In this study, we aimed to validate these findings in a younger cohort with ASD (aged 2-4 years) and assess their potential use as biomarkers for the early diagnosis of ASD. A total of 108 young children aged 2-4 years were recruited, including 66 children with ASD and 42 age- and sex-matched controls. Using next-generation sequencing and advanced bioinformatics, we validated the differential expression of 17 miRNAs in ASD, which showed consistent dysregulation across both the current and previous cohorts. We also observed significant correlations between several miRNAs and participants' age, suggesting that age is a key factor influencing dynamic miRNA changes, particularly in the ASD group. Pathway analysis linked these miRNAs to critical regulatory networks involved in neurodevelopment and immune responses. Finally, we found that a combination of four miRNAs (miR-4433b-5p, miR-15a-5p, miR-335-5p, and miR-1180-3p) exhibited high diagnostic accuracy, with an area under the curve (ROC-AUC) of 0.936 (95% CI = 0.892, 0.980