Chronic kidney disease (CKD) is a progressively worsening condition that erodes renal function over time, reduces quality of life, and can ultimately culminate in kidney failure with far-reaching systemic complications. In addition to reduced filtration, worsening kidney function disrupts mineral homeostasis and leads to CKD–mineral and bone disorder (CKD-MBD). Dysregulated calcium handling and maladaptive endocrine responses contribute to bone pathology and increase cardiovascular calcification risk; therefore, serial calcium monitoring remains clinically relevant for longitudinal CKD management. Conventional calcium measurements are typically obtained with centralized analyzers or laboratory assays (e.g., colorimetry and electrode/optical readouts). Despite high accuracy, the required instrumentation, controlled operating conditions, and pretreatment steps complicate rapid point-of-care deployment, especially when only microliter-scale biofluids are available. Accordingly, this study develops a finger-actuated microfluidic colorimetric platform capable of determining calcium ion concentrations in human biofluids, such as whole blood, serum, and urine. The platform integrates a three-dimensional PMMA/paper microchip with a compact reader that maintains stable temperature control while enabling CMOS-based optical detection. With just 6 μL of sample, a brief finger press propels the biofluid across an internal filtration layer, generating serum or cleaned urine that subsequently reacts with a pre-deposited murexide reagent. Under optimized conditions (1.6% reagent, 50°C, 3 min), the signal follows a strong logarithmic relationship with calcium concentration (Y = 47.273 ln X + 28.890; R² = 0.9905), supporting quantification over 1–40 mg/dL and a detection limit of 0.2 mg/dL. Across 80 clinical CKD specimens spanning serum, whole blood, and urine, results aligned closely with the NM-BAPTA reference assay, with R² values exceeding 0.97.