Neuroblastoma is one of the most common cancers of childhood, and early detection as well as efficient treatment options are crucial for patient survival. This study describes the detection of potential novel markers of neuroblastoma via utilization of serum-derived autoantibodies. It also shows that autoantibodies against the Y-box binding protein 1 (YB-1) are elevated in serum from neuroblastoma patients, with highest abundance in serum from stage 4 patients. YB-1 is detectable in medium conditioned by neuroblastoma cell lines, most probably due to active secretion. The extracellular presence of YB-1 in the area surrounding the tumor would expose the protein to the immune system, and might trigger the formation of autoantibodies. Knockdown of the YB-1 protein in neuroblastoma cells increased DNA double-strand breaks and reduced cell viability. Co-immunoprecipitation experiments also showed that YB-1 was associated with proteins involved in various repair pathways, Ku80, Rad51 and XPC, in neuroblastoma cells. YB-1 was shown to be a direct target of MYCN. Amplification of the MYCN gene is the strongest predictor of bad prognosis for neuroblastoma known to date, and the fact that YB-1 is a MYCN target indicates that YB-1 could be an important factor in the development and progression of neuroblastoma. Additionally the inhibition of YB-1 as a new downstream target of Myc-proteins may provide new insights into the carcinogenesis and possible therapy of MYCN-amplified tumors. Neuroblastoma is a very heterogeneous disease and early detection as well as consequent patient monitoring and staging is crucial to prevent patient over- or undertreatment. Results of this study may help with tumor staging and YB-1 may be a possible future target for treatment of resistant tumors.