In this paper, we report, for the first time, the use of a smartphone to image and quantify biochemiluminescence coupled biospecific enzymatic reactions to detect analytes in biological fluids. Using low-cost three-dimensional (3D) printing technology, we fabricated a smartphone accessory and a minicartridge for hosting biospecific reactions. As a proof-of-principle, we report two assays: a bioluminescence assay for total bile acids using 3α-hydroxyl steroid dehydrogenase coimmobilized with bacterial luciferase system and a chemiluminescence assay for total cholesterol using cholesterol esterase/cholesterol oxidase coupled with the luminol-H2O2-horseradish peroxidase system. These assays can be performed within 3 min in a very straightforward manner and provided adequate analytical performance for the analysis of total cholesterol in serum (limit of detection (LOD) = 20 mg/dL) and total bile acid in serum and oral fluid (LOD = 0.5 μmol/L) with a reasonable accuracy and precision. Smartphone-based biochemiluminescence detection could be thus applied to a variety of clinical chemistry assays.