We developed fibroblast activation protein alpha(FAP alpha)-sensitive magnetic iron oxide nanoparticles (MNPs) by conjugating a substrate-reporter tandem peptide as a synthetic biomarker to the surface of MNPs (marker-MNPs). In vitro, the marker-MNPs showed stability when treated with serum or urine and exhibited high susceptibility and specificity for FAPa enzyme and 3T3/FAP alpha cell line. Furthermore, the marker-MNPs were administered to esophageal squamous cell carcinoma xenograft tumor mice; they reached the tumor tissues in the mice, where they were cleaved effectively by the local overexpressed FAP alpha to release the reporter peptide and filter it into the urine. The tumor targeting and biodistribution of marker-MNPs were verified by in vivo imaging. The cleaved reporter peptides in urine detected by enzyme-linked immunosorbent assay have high diagnostic accuracy for esophageal squamous cell carcinoma (area under the receiver-operating characteristic curve = 1.0). Our study implies a promising strategy of utilizing the low-cost and noninvasive synthetic urinary probe-coated nanoparticles for the diagnosis of FAPa-positive solid tumors, except for in renal cancer.