Appendicitis is inflammation and swelling of the appendix that can occur at any age. It can be caused by fecalith blocking the appendix entrance and has been associated with lamina propria thickening due to infection. Some reports have observed a relation between appendicitis and hyperplastic lymphoid tissue, which might be caused by infections like adenovirus, while others have linked adenovirus infection and appendicitis in children. This study retrospectively analyzed appendix tissue pathology from children who underwent appendectomy and more directly investigate the relation between adenovirus infection and appendicitis. We included 120 children under age 18 who underwent appendectomy in patients with appendicitis at Cathay General Hospital between January 2016 and January 2021. Adenovirus immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR) were analyzed to determine the positive rate of adenovirus in appendicitis. Thirty-one cases (25.8%) were positive for anti-adenovirus antibody IHC. qPCR was positive for adenovirus in 42 cases (35%). Eleven (9.2%) tested positive by both IHC and qPCR. Our pathological evidence directly confirms a relation between adenovirus infection and appendicitis. IHC and qPCR analyses confirmed evidence of adenovirus infection in patients with appendicitis. qPCR is as useful and reliable as IHC, and more sensitive, for diagnosing adenovirus in appendicitis.