Targeted therapy opened a new era for the treatment of malignancies. Recently, there is growing interest in the development of novel targeted ligands except for monoclonal antibodies (mAbs). Aptamers are a class of therapeutic oligonucleotides form specific three dimensional structures dictated by their sequences. Aptamers can bind to a wide range of targets with high sensitivity and specificity, making it an ideal candidate for disease diagnosis and therapy. Herein, an anti-CD20 DNA aptamer (ACDA) was successfully screened from Harvard library by systematic evolution of ligands by exponential enrichment (SELEX). Experimental results demonstrated that ACDA can bind to surface CD20 than Rituximab Fab fragments with stronger binding affinity. With mass arming ACDA to a long chain polyethyleneimine (PEI) polymer, the resultant aptamer–polymer conjugates (P-ACDA) can induce potent caspase dependent apoptosis in targeting Non-Hodgkin's Lymphoma (NHL) cells via the crosslink of cellular CD20. All the results indicated that ACDA not only can be developed as targeting ligands for specifically delivering diagnostic or therapeutic drugs, but also can themselves be effective therapeutic candidates in treating NHL, which deserves for further investigation in the clinic.