In this work, a highly sensitive ultrahigh-performance liquid chromatography and quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) method has been developed and validated by the quantifications of danshensu (DSS) and salvianolic acid B (SAB) in Danhong injection (DHI) in human plasma, urine and feces for the pharmacokinetic and excretion studies. The DSS and SAB were extracted from biological matrix by direct protein precipitation with fivefold volume of methanol and separated by a Waters CORTECS C18+ chromatographic column (100 × 2.1 mm, 2.7 μm) with gradient elution (mobile phase: methanol–H2O, both containing 0.1% formic acid). The detection of analytes was achieved using negative ion ESI in Full MS/dd-MS2 (data-dependent MS2) scan mode. The human subjects received either a single intravenous injection (i.v.) dose (containing 0.331 mg/kg DSS and 0.117 mg/kg SAB) or a single intramuscular injection (i.m.) dose (containing 0.663 mg/kg DSS and 0.234 mg/kg SAB) of DHI. For i.v., the Cmax, half-life (t1/2) and AUC0−∞ were 906.2 ± 31.82 μg/L, 0.604 ± 0.103 h and 242.19 ± 18.24 μg/L × h for DSS and 1118.5 ± 35.64 μg/L, 0.893 ± 0.159 h and 277.63 ± 11.24 μg/L × h for SAB, respectively. For i.m., the Cmax, Tmax, t1/2 and AUC0−∞ were 243.0 ± 29.1 μg/L, 0.67 h, 0.71 ± 0.03 h and 318.98 ± 7.62 μg/L × h for DSS and 119.4 ± 6.51 μg/L, 0.50 h, 1.08 ± 0.18 h and 185.63 ± 4.23 μg/L × h for SAB, respectively. Based on the AUC0−∞ obtained from i.m. and i.v. administrations, the absolute bioavailabilities (F) were estimated to be 65.9% for DSS and 33.4% for SAB.