Fluorescence spectra of biphenylene vapor were observed under collision-free conditions with excitation into different vibrational levels of the lowest excited singlet state (S1) and of the second excited singlet state (S2). The fluorescence spectrum resulting from excitation into the vibrational level of S1 shows a distinct structure, while the one from excitation into the vibrational level of S2 is diffuse and slightly red-shifted, indicating that the fluorescence is emitted from a vibrational level of S1 even when the biphenylene molecule is excited to S2. The fluorescence yield is as low as 3.3 × 10-4 at the highest, and it decrease with increasing excitation energy. The unusually low fluorescence quantum yield of biphenylene, compared with those of other alternant hydrocarbons, is attributed to fast S1 → S0 internal conversion caused by a large configurational difference between S1 and S0. The vibronic coupling is discussed which allows the forbidden S1 ↔ S0 transition to occur.