Streptococcus pneumoniae (SP) is a pathogenic bacterium and a major cause of community-acquired pneumonia that could be fatal if left untreated. Therefore, rapid and sensitive detection of SP is crucial to enable targeted treatment during SP infections. In this study, DNA tetrahedron (DNA TH) with a hollow structure is anchored on gold electrodes to construct an electrochemical immunosensor for rapid detection of pneumococcal surface protein A (PspA) peptide and SP lysate from synthetic and actual human samples. This DNA nanostructure-based immunosensor displays excellent electrochemical activity toward PspA with a sensitive linear region from 0 to 8 ng/mL of PspA peptide and a low limit of detection (LOD) of 0.218 ng/mL. In addition, this DNA-TH-based immunosensor exhibits good sensing performance toward SP lysate in a clinically relevant linear range from 5 to 100 CFU/mL with a LOD of 0.093 CFU/mL. Along with these attractive features, this electrochemical immunosensor is able to specifically recognize and detect the PspA peptide mixed with other physiologically relevant components like bovine serum albumin (BSA) and lipopolysaccharide. In addition, our sensor could detect SP lysate even when dispersed in BSA or Escherichia coli lysate. Lastly, uncultured samples from the nasal cavity, mouth, and axilla of a human subject could be successfully determined by this well-designed electrochemical immunosensor.