Bladder cancer is the fourth most common cancer in men, and it is becoming a prevalent malignancy. Most of the regular clinical examinations are prompt evaluations with cystoscopy, renal function testing, which require high-precision instrument, well-trained operators, and high cost. In this study, a microfluidic paper-based analytical device (mu PAD) was fabricated to detect nuclear matrix protein 22 (NMP22) and bladder cancer antigen (BTA) from the urine samples. Urine samples were collected from 11 bladder cancer patients and 10 well-beings as experiment and control groups, respectively, to verify the working efficiency of mu PAD. A remarkable checkout efficiency of up to 90.91% was found from the results. Meanwhile, this method is feasible for home-based self-detection from urine samples within 10 min for the total process, which provides a new way for quick, economical, and convenient tumor diagnosis, prognosis evaluation, and drug response.