Paper-based microfluidic analysis devices (μPADs) have attracted attention as a cost-effective platform for point-of-care testing (POCT), food safety, and environmental monitoring. Recently, three-dimensional (3D)-μPADs have been developed to improve the performance of μPADs. For accurate diagnosis of diseases, however, 3D-μPADs need to be developed to simultaneously detect multiple biomarkers. Here, we report a 3D-μPADs platform for the detection of multiple biomarkers that can be analyzed and diagnosed with a smartphone. The 3D-μPADs were fabricated using a 3D digital light processing printer and consisted of a sample reservoir (300 µL) connected to 24 detection zones (of 4 mm in diameter) through 8 microchannels (of 2 mm in width). With the smartphone application, eight different biomarkers related to various diseases were detectable in concentrations ranging from normal to abnormal conditions: glucose (0–20 mmol/L), cholesterol (0–10 mmol/L), albumin (0–7 g/dL), alkaline phosphatase (0–800 U/L), creatinine (0–500 µmol/L), aspartate aminotransferase (0–800 U/L), alanine aminotransferase (0–1000 U/L), and urea nitrogen (0–7.2 mmol/L). These results suggest that 3D-µPADs can be used as a POCT platform for simultaneous detection of multiple biomarkers.