​Chemotherapy continues to be a fundamental approach in treating various cancers, with combination therapies frequently improving effectiveness while minimizing toxicity. Recently, the therapeutic potential of combining Paclitaxel and Leucovorin drugs has been highlighted in cancer treatment. However, both agents pose challenges in terms of toxicity and necessitate careful dosing and monitoring. To address this, we report the selection of two ssDNA aptamers and their use in the electrochemical biosensing of Paclitaxel and Leucovorin. Using the SELEX process, five sequences were generated for paclitaxel, labeled P1 to P5, while four aptamers for leucovorin were identified (L1 to L4). Based on affinity studies, the aptamers P3 and L1 exhibiting the lowest dissociation constants, were selected for the design of Paclitaxel and Leucovorin biosensors, respectively. The developed aptasensors were applicable within the ranges of 10–1000 pg/mL and 3–500 pg/mL with the low detection limits of 0.02 and 0.0077 pg/mL for Paclitaxel and Leucovorin, respectively. A good selectivity was also attained against different drugs, including chemotherapeutic compounds. Finally, real samples analysis was also carried out showing good recovery rates ranging from 91.3% to 109% with RSDs lower than 5%. In comparison to traditional techniques, this platform shows high performance and is user-friendly, presenting a novel and efficient approach for monitoring paclitaxel and leucovorin in chemotherapy regimens.​​