Mass spectrometry-based quantitative proteomics, consisting of relative and absolute parts, has been used to discover and validate proteins with key functions related to physiological and pathological processes. Currently, stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) is the most commonly used method for the absolute determination of proteins in a biological sample. A prerequisite for this method is obtaining internal standards with isotope labels. Although many approaches have been developed for the labeling and preparation of internal peptides, expensive stable isotope labeling coupled with SID-MRM-MS has limited the application and development of an absolute quantitative method. Recently, a low-cost strategy using metal–tag labeling and MS has been developed for relative quantification of peptides or proteins. The introduction of labeling using metal tags has the merits of allowing multiple labeling and enlarging the mass shift to overcome the overlap of adjacent isotope clusters. However, most papers described MRM-MS for protein absolute quantification based on the metal in its peptides labelled with metal by inductively coupled plasma mass spectrometry (ICP MS) but not on its peptides labelled with metal. In this work, a novel approach based on metal–tag labeling coupled with MRM-MS was established for the absolute quantification of peptides or proteins. The principle of the method is that a bifunctional chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid bearing an N-hydroxysuccinimide ester (DOTA-NHS ester), is used to modify the N-termini of signature peptides from a target protein, and the modified peptides then chelate a certain metal, such as thulium, to form metal-tagged peptides (Tm–DOTA–P). Internal peptides are chemically synthesized and labeled with another metal, such as terbium (Tb–DOTA–P), as the internal standard. Both the Tb–DOTA- and Tm–DOTA-labeled peptides in samples can be analysed via MRM-MS. The experimental results show that the accuracy (%RE) and precision (%RSD) of the approach are both below 15%, and the lower limit of quantification (LOQ) is 0.8 fmol μL−1, with good linearity (R2 > 0.99) observed covering the range of 2 orders of magnitude. Furthermore, one protein, enolase, in an extract from Thermoanaerobacter tengcongensis was successfully quantified, which demonstrates that this novel absolute quantification method is a new strategy for simple, rapid, low-cost and accurate absolute protein quantification in a complex biological sample.