Telomerase and poly(ADP-ribose) polymerase-1 (PARP-1) are two potential cancer biomarkers and are closely related to tumor initiation and malignant progression. TOTO-1 is well-known for differentiating ss-DNA from ds-DNA because it is virtually non-fluorescent without DNA and exhibits very low fluorescence with ss-DNA, while it emits strong fluorescence with ds-DNA. In this paper, for the first time, it was found that TOTO-1 has high fluorescence selectivity and sensitivity towards the G bases in single-stranded DNA and poly(ADP-ribose) (PAR). Poly(dG) was used as the model target to explore its possible mechanism. Molecular dynamics (MD) simulation proved that intramolecular π-π stacking existed in TOTO-1 (in an aqueous solution), while intermolecular π-π stacking formed between TOTO-1 and poly(dG) in a similar way as that observed for dsDNA. Interestingly, telomerase and PARP-1 catalyzed the formation of G-rich DNA and PAR in vivo, respectively. Therefore, TOTO-1 was explored in detecting both of them, obtaining satisfactory results. To the best of our knowledge, no probe has been reported to recognize PAR. It is also the first time where telomerase is detected based on the specific recognition of G bases. Importantly, integrating multiple functions into one probe that can detect not only telomerase but also PARP-1 will significantly raise the specificity of screening cancer and decrease false positive proportion, which make TOTO-1 a promising candidate probe for clinical diagnosis and pharmaceutical screening.