Direct quantitative analysis of low-abundance protein biomarkers by electrospray ionization mass spectrometry (ESI-MS) remains challenging due to poor ionization efficiency and matrix interferences. Herein, we report an ultrasensitive analytical platform, termed CRISPR/Cas12a-mediated liposomal amplification coupled with electrospray ionization mass spectrometry (CMLA-MS), that overcomes this limitation by integrating CRISPR/Cas12a-mediated dual-cascade signal amplification with an ESI-MS readout. The strategy converts the detection of poorly ionizable protein molecules into the quantification of numerous, highly ionizable small-molecule reporters: proteins trigger Cas12a trans-cleavage (first amplification), which subsequently cleaves single-stranded DNA (ssDNA) probes anchored to signal-loaded liposomes, causing the burst release of thousands of MS-detectable reporters (second, physical amplification). This dual-amplification strategy enabled an exceptionally low limit of detection (LOD) of 10.8 fg/mL, and the method successfully quantified cardiac troponin I (cTnI) in clinical serum samples with high recoveries (90.3–101.6%).