摘要： The development of new rapid and reliable detection technology of Cryptosporidium, a deadly waterborne pathogen, using a three-dimensional micro total analysis systems (3D μTAS) is the main goal of this work. An approach to enhance DNA detection on a patterned configurable 3D μTAS electrochemical DNA biosensor to human genotype of Cryptosporidium was successfully developed in present study. A novel 3D μTAS, comprising three layers, was fabricated to achieve better result comparing the commercial SPE. The first layer was consisted a circular gold coated area (gold dot) in the center of a PI sheet and the third layer was consisted of a common homemade screen printed electrode (SPE). The second layer was included PDMS channels and reservoir for sample delivery. The biosensor was fabricated on the gold dot of the first layer and electrochemical signal was recorded using the differential pulse voltammetry (DPV) and impedance methods on the third layer and the results were compared with biosensor fabricated on commercial SPE. The calibration curve of 3D μTAS DNA biosensor shows a detection limit of 1.8 ng/mL and a sensitivity of 12.844 μA/(μg mL-1) in a linear range of 2.5 ng/mL to 0.1 μg/mL, which are 10 times higher detection limit and 23 higher sensitivity of commercial SPE in a similar linear range. The specificity of prepared DNA sensor was conducted by using non-complementary, single, three and five mismatches strands and the potential of the biosensor in discriminating single mismatch detection was experimentally appraised.