The occurrence of cancers is often accompanied by the abnormal expression of several sorts of biomarkers (e.g., nucleic acids and proteins). The multiplex assessment of them would substantially aid in the early detection and precise diagnosis, which is often hampered by their different detection schemes, different reaction matrix and reagents, and spectral overlapping. Herein, we propose a simple and sensitive mass spectrometric method for the multiplex detection of nucleic acid and protein, in which liver cancer-related biomarkers miRNA 223 and alpha-fetoprotein (AFP) were selected as model analytes. The self-amplification effect of metal atom-based nanoparticle probes can provide high sensitivity in complex serum samples without any additional amplification procedure. The detection limits for the simultaneous detection of miRNA 223 and AFP were 103 (2.1 pM) and 219 amol (0.15 ng/mL), respectively, with high specificity and selectivity. The proposed method is potentially useful for the rapid screening of cancers.