Phosphatidylserine (PS) is a phospholipid that is an important component of all cell membranes. Normally, phospholipid PS is only found on the cytosolic surface of the plasma membrane. Obligatory translocation of PS from the interior to the exterior side of the plasma membranes is a potential biomarker for apoptotic cell death. The SELEX technology (Systematic Evolution of Ligands by Exponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers. In this study, entropic fragment based approach (EFBA) was used to design a set of DNA aptamers bind specifically with PS based on seed-and-grow strategy. Each member of designed short-sequence DNA aptamers consists of a 6 nucleotides while long-sequence DNA aptamers consists of an 11 nucleotides. The binding properties of the designed DNA aptamers with phospholipid PS have been studied using NanoDrop without fluorescent tags attached to them. This technique has been proposed to measure concentrations of relative PS bound aptamers versus unbound ones and to analyze the phospholipid binding properties of aptamers. The Short-sequence DNA aptamers show stronger binding affinity for phospholipid PS compared with long ones. Also short-sequence DNA aptamer (AAAGAC) has the highest binding affinity compared with the other short ones and also the other two long ones. These results suggest that the phospholipid PS binding of DNA aptamers is very much sequence specific and depends also on the number of nucleotides in the DNA aptamers. The present results thus disclose a novel assay for phospholipid PS recognition in apoptotic cells, which may open up the possibility of discovering aptamer based diagnostic tools to be used in treatment of many diseases including cancer.