ObjectivesAptamer sgc8c is a short DNA sequence that can target protein tyrosine kinase 7 (PTK7), which was overexpressed on many tumour cells. This study aimed to fabricate a novelty DNA nanostructure drug delivery system target on PTK7‐positive cells—CCRF‐CEM (human T‐cell ALL).MethodsAptamer‐modified tetrahedron DNA was synthesized through one‐step thermal annealing process. The sgc8c‐TDNs (s‐TDNs) loading DOX complexes were applied to investigate the effect to PTK7‐negative and ‐positive cells.ResultsWhen s‐TDN:DOX acted on PTK7‐positive and ‐negative cells respectively, the complexes exhibited specific toxic effect on PTK7‐positive cells but not on PTK7‐negative Ramos cells in vitro research.ConclusionsIn this work, we successfully constructed a PTK7‐targeting aptamer‐guided DNA tetrahedral nanostructure (s‐TDN) as a drug delivery system via a facile one‐pot synthesis method. The results showed that s‐TDN:DOX exhibited enhanced cytotoxicity against PTK7‐positive CCRF‐CEM cells, with a minor effect against PTK7‐negative Ramos cells. Hence, this functionalized TDNs drug delivery system displayed its potential application in targeting PTK7‐positive tumour T‐cell acute lymphoblastic leukaemia.