Opioid peptides released during digestion of dietary proteins such as casein, were suggested to contribute to autism development, leading to the announcement of opioid excess hypothesis of autism. This paper examines role of enzyme proline dipeptidyl peptidase-4 (DPPIV; EC 188.8.131.52) and it is exogenous substrate, β-casomorphin-7 (BCM7) in autism etiology. Our study included measurements of DPPIV and BCM7 concentrations in serum and urine, which were analyzed with ELISA assays and activity of DPPIV was measured by colorimetric test. The effect of opioid peptides from hydrolysed bovine milk on DPPIV gene expression in peripheral blood mononuclear cells (PBMC) in autistic and healthy children was determined using the Real-Time PCR (Polymerase Chain Reaction) method. Our research included 51 healthy children and 86 children diagnosed with autism spectrum disorder (ASD, ICDF84). We determined that the concentration of BCM7 in serum was significantly, 1.6-fold, higher in the ASD group than in controls (p < 0.0001). Concentration of DPPIV was found to also be significantly higher in serum from ASD children compared to the control group (p < 0.01), while we did not notice significant difference in enzymatic activity of serum DPPIV between the two study groups. We confirmed correlation according to the gender between analyzed parameters. The inspiration for this study emanated from clinical experience of the daily diet role in relieving the symptoms of autism. Despite this, we have concluded that milk-derived opioid peptides and DPPIV are potentially factors in determining the pathogenesis of autism; conducted studies are still limited and require further research.