β-CD-HMTA-L-Tyr complex, formed in the host guest inclusion reaction carried out between host molecule β-cyclodextrin (β-CD) in β-CD-HMTA (HMTA is methenamine) and guest molecule L-tryptophan (L-Tyr), possessing the characteristic of room temperature phosphorescence (RTP). Bovine serum albumin (BSA) reacted with L-Tyr to form a complex of cage structure bringing in the sharply RTP signal quenching of L-Tyr. Based on the above facts, a new ultra-sensitive solid substrate room temperature phosphorimetry (SSRTP) for the determination of trace protein has been established using β-CD-HMTA-L-Tyr complex as a phosphorescence probe. Under the optimum conditions, the linear range of this method was 0.0040-0.56 agspot(-1) with a detection limit (D.L.) as 0.92 zgspot(-1), and the regression equations of working curve was ΔI(p)=0.8239+162.5 m(BSA) (agspot(-1), n=8) with the correlation coefficient (r) of 0.9994. The relatively standard deviation (RSD) and the recovery of SSRTP were 4.8-3.3% and 96.7-102%, respectively, indicating that this method had good repeatability. The proposed phosphorescence probe has been applied in the detection of protein in real samples and the results agreed well with those obtained with SSRTP using methylene blue-sodium tetraphenylborate as phosphorescence probe. Meanwhile, the reaction mechanism for the determination of trace protein with β-CD-HMTA-L-Tyr complex as phosphorescence probe has been discussed.