This paper presents a novel method for direct detection of Plasmodium falciparum histidine rich protein-2 (PfHRP-2) antigen using carbon nanofiber (CNF) forests grown on glass microballoons (NMBs). Secondary antibodies specific to PfHRP-2 densely attached to the CNFs exhibit extraordinary ability for the detection of minute concentrations of Plasmodium species. A sandwich immunoassay protocol was employed, where a glass substrate was used to immobilize primary antibodies at designated capture zones. High signal amplification was obtained in both colorimetric and electrical measurements due to the CNFs through specific binding. As a result, it was possible to detect PfHRP-2 levels as low as 0.025 ng/mL concentration in phosphate buffered saline (PBS) using a visual signal within only 1 min of test duration. Lower limits of 0.01 ng/mL was obtained by measuring the electrical resistivity of the capture zone. This method is also highly selective and specific in identifying PfHRP-2 and other Plasmodium species from the same solution. In addition, the stability of the labeling mechanism eliminates the false signals generated by the use of dyes in current malaria rapid diagnostic test kits (MRDTs). Thus, the rapid, sensitive and high signal amplification capabilities of NMBs is a promising tool for early diagnosis of malaria and other infectious diseases.