The human genome encodes thousands of natural antisense long noncoding RNAs (lncRNAs); they play the essential role in regulation of gene expression at multiple levels, including replication, transcription and translation. Dysregulation of antisense lncRNAs plays indispensable roles in numerous biological progress, such as tumour progression, metastasis and resistance to therapeutic agents. To date, there have been several studies analysing antisense lncRNAs expression profiles in cancer, but not enough to highlight the complexity of the disease. In this study, we investigated the expression patterns of antisense lncRNAs from osteosarcoma and healthy bone samples (24 tumour -16 bone samples) using RNA sequencing. We identified 15 antisense lncRNAs (RUSC1-AS1, TBX2-AS1, PTOV1-AS1, UBE2D3-AS1, ERCC8-AS1, ZMIZ1-AS1, RNF144A-AS1, RDH10-AS1, TRG-AS1, GSN-AS1, HMGA2-AS1, ZNF528-AS1, OTUD6B-AS1, COX10-AS1 and SLC16A1-AS1) that were upregulated in tumour samples compared to bone sample controls. Further, we performed real-time polymerase chain reaction (RT-qPCR) to validate the expressions of the antisense lncRNAs in 8 different osteosarcoma cell lines (SaOS-2, G-292, HOS, U2-OS, 143B, SJSA-1, MG-63, and MNNG/HOS) compared to hFOB (human osteoblast cell line). These differentially expressed IncRNAs are potential therapeutic targets and biomarkers for osteosarcoma.