SIGNIFICANCE: Intravascular near-infrared fluorescence (NIRF) imaging aims to improve the inspection of vascular pathology using fluorescent agents with specificity to vascular disease biomarkers. The method has been developed to operate in tandem with an anatomical modality, such as intravascular ultrasound (IVUS), and complements anatomical readings with pathophysiological contrast, enhancing the information obtained from the hybrid examination. AIM: However, attenuation of NIRF signals by blood challenges NIRF quantification. We propose a new method for attenuation correction in NIRF intravascular imaging based on a fluorophore-coated guidewire that is used as a reference for the fluorescence measurement and provides a real-time measurement of blood attenuation during the NIRF examination. APPROACH: We examine the performance of the method in a porcine coronary artery ex vivo and phantoms using a 3.2F NIRF-IVUS catheter. RESULTS: We demonstrate marked improvement over uncorrected signals of up to 4.5-fold and errors of < 11 % for target signals acquired at distances up to 1 mm from the catheter system employed. CONCLUSIONS: The method offers a potential means of improving the accuracy of intravascular NIRF imaging under in vivo conditions.