The sensitive analysis of DNA is of extreme importance in the field of clinical diagnosis, targeted therapeutics, gene therapy, and a variety of biomedical studies. Even though significant achievements have been made in the detection of single-stranded DNA (ssDNA), facile strategy for the detection of double-stranded DNA (dsDNA) is still lacking. In this study, we have constructed an electrochemical DNA sensor based on homogeneous hybridization. Target dsDNA is thermal denatured and hybridized with thiolated capture probe and biotin-labeled reporter probe in solution phase. Excessive amount of the probes and precise control of hybridization temperature ensure the predominant formation of sandwich SH-dsDNA-biotin rather than reannealing product of the target dsDNA. A bovine serum albumin (BSA)-modified gold surface is designed to improve self-assembly capability of the sandwich SH-dsDNA-biotin structure in the presence of excessive SH-ssDNA probe. Thus, the target dsDNA can be successfully detected by using enzyme-linked amperometric amplification. By employing this strategy, dsDNA can be detected in a linear range from 5 pM to 1 nM with a detection limit down to 800 fM (S/N = 3). Cryptococcus neoformans in clinical real samples from cryptococcal meningitis patients can be discriminated successfully by the proposed method. Our research would make the electrochemical biosensor be an excellent candidate for pathogens detection in clinical diagnosis and prognosis.