Singlet oxygen (1O2) is widely recognized as the primary cytotoxic agent during photodynamic therapy (PDT). The quantitation of 1O2 generation during PDT plays an important role for 1O2 based PDT dosimetry. However, it is still a challenging task for direct detection of 1O2 using an optical system due to its extremely weak luminescence at 1270 nm. In this paper, a highly sensitive optical fiber detection system was developed to measure the time-resolved 1O2 luminescence spectra from two model photosensitizers (Rose Bengal and TMPyP) at various concentrations (1.25, 2.5, 5.0, and 10.0 μM), The 1O2 luminescence signal was excited by a diode-pumped, Q-switched, frequency-doubled 523- nm Nd:YLF laser and collected by an optical fiber probe coupled with a highly sensitive NIR photomultiplier tube (PMT). Experimental results indicate that the 1O2 luminescence intensity shows a linear enhancement both with the increase of concentrations of Rose Bengal and TMPyP. The 1O2 luminescence signal dramatically decreased after the addition of 50 mM sodium azide, a specific 1O2 quencher. Furthermore, the signal-to-noise ratio (SNR), calculated under the condition of 10 mM Rose Bengal solution, is higher than those obtained from optical fiber 1O2 luminescence detection systems based on InGaAs/InP single photon avalanche diode and superconducting nanowire single-photon detector. Our results suggest that the home-built optical fiber system with a high sensitivity for 1O2 luminescence detection will have a great potential for clinical applications in PDT dosimetry.