Human Pituitary Tumour Transforming Gene 1 (PTTG1) is an oncoprotein involved in maintaining chromosome stability and acts as a biomarker for a panel of cancers. In this study, we endeavoured to generate an RNA aptamer against PTTG1. The RNA aptamer, SECURA-3 has an estimated equilibrium dissociation constant of 16.41 ± 6.4 nM. The aptamer was successfully harnessed in several diagnostic platforms including ELASA, aptamer-based dot blot and aptamer-based western blot. SECURA-3 was also unveiled as a potential probe that could replace its counterpart antibody in the histostaining-based detection of PTTG1 in HeLa and MCF-7 formalin-fixed paraffin-embedded cell blocks. In the aspect of therapeutics, SECURA-3 RNA aptamer demonstrates an antagonistic effect by antagonizing the interaction between PTTG1 and CXCR2, as revealed in the in vitro competitive nitrocellulose filter binding assay and dual-luciferase reporter assay in HeLa cells. As the first anti-PTTG1 aptamer, SECURA-3 RNA aptamer has immense diagnostic and therapeutic properties.