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Retinoblastoma (RB) is the most common paediatric intraocular tumour. The management of RB has improved the survival and vision with recent advances in the treatment. Improved therapeutic approaches focussing on targeting tumours and minimizing the treatment-associated side effects are being developed. In this study, we generated a ssDNA aptamer against RB by cell-SELEX and high-throughput sequencing using Weri-RB1 cell line as the target, and Muller glial cell line Mio-M1 as the control. Three aptamers were selected based on the number of repetitions in NGS and phylogenetic relationship and evaluated by flow cytometry to assess their binding affinity and selectivity. The dissociation constant, Kd values of three selected aptamers were found to be in the nanomolar range. Aptamer VRF-CSRB-01 with the best binding affinity and a Kd value of 49.41 ± 7.87 nM was further characterized. The proteinase and temperature treatment indicated that VRF-CSRB-01 targets surface proteins, and has a good binding affinity and excellent selectivity under physiological conditions. The aptamer VRF-CSRB-01 was stable over 72 h in serum and 96 h in cerebral spinal fluid and vitreous. With the high affinity, specificity, stability and specific recognition of clinical RB tumours, VRF-CSRB-01 aptamer holds potential for application in diagnosis and targeting RB. [ABSTRACT FROM AUTHOR]