Neuroblastoma (NB) represents a heterogeneous pediatric cancer with diverse clinical behaviours influenced by genetic alterations such as MYCN amplification and 11q chromosomal deletions. This study explores the role of microRNAs (miRNAs), specifically hsa-miR-34b and hsa-miR-34c, in neuroblastoma pathogenesis across multiple cell lines such as NB69, SH-SY-5Y, IMR32, SK-N-BE, and SK-N-AS, each characterized by distinct genetic backgrounds. These miRNAs regulate critical pathways in NB, including cell cycle progression, apoptosis, and oncogene expression. Using quantitative PCR (qPCR) and U6B small nuclear RNA (RNU6B) as endogenous control, miRNA expression levels were assessed across three passages of these cell lines. Only NB69 and IMR32 showed detectable expression of these two miRNAs, while SH-SY-5Y, SK-N-AS, and SK-N-BE exhibited no detectable levels. Notably, the 11q deletion in SK-N-AS may have contributed to the loss of these miRNAs. In SH-SY-5Y cells, the absence of detectable hsa-miR-34b and hsa-miR-34c expression may be influenced by epigenetic regulation, such as DNA methylation. In SK-N-BE, MYCN amplification likely suppresses miRNA expression, consistent with its role in inhibiting tumor-suppressor miRNAs. Statistical analysis revealed significantly lower expression of both miRNAs in the MYCN-amplified IMR32 cell line compared to the NB69 cell line lacking both MYCN amplification and 11q deletion. The variability observed across cell lines highlights the complex regulation of these miRNAs in NB and underscores the need for further research to establish their potential as biomarkers or therapeutic targets.