A definitive test that is universally accepted for accurately diagnosing Autism Spectrum Disorders (ASD) has yet to be developed. Because ASD results from a combination of genetic susceptibility and environmental factors such as infections, dietary proteins, xenobiotics, and gut dysbiosis, we measured biomarkers in the blood of healthy controls and patients with ASD and Crohn’s disease. Using ELISA methodologies, we first measured antibodies against gluten and non-gluten proteins of wheat, alpha-and beta-casein, heavy metals (ethyl mercury, aluminum hydroxide, aluminum phosphate), bisphenol A, artificial food colorants, and glyphosate. These chemicals or their metabolites can affect the gut microbiome and induce overproduction of bacterial toxins, which may result in barrier breakdown. Therefore, we also measured antibodies against LPS, bacterial cytotoxins, occludin/zonulin, and vinculin. Furthermore, since bacterial toxins can damage both the gut barrier and blood-brain barrier, we also measured antibodies against claudin-5, S100B, glial fibrillary acidic protein, myelin basic protein, cerebellar peptide and synapsin. Compared to controls, ASD groups had much higher levels of antibodies against food antigens, chemicals that form neo-antigens with human tissue, tight junction proteins, and neural cells. Regarding cellular immunology, we also measured pro-inflammatory cytokines, including IL-1 beta, TNF-alpha, IL-6, and IL-17; all were found to be significantly elevated in ASD patients. Finally, the regulatory cytokines such as IL-10, TGF-beta and regulatory T cells were measured in comparison with controls and were found to be lower in subjects with ASD. The results of this study indicate that a dysregulated immune system, including overproduction of antibodies and inflammatory cytokines with down-regulation of regulatory T cells, plays a role in autism. The development of biomarkers such as those used in this study is essential for improving the diagnosis and management of ASD.