Abstract <p>Putrescine, a vital polyamine involved in various cellular functions, particularly cell proliferation, is often associated with health conditions such as cancer. Traditional methods for putrescine determination suffer from limitations such as high detection limits and complexity. Hence, there is a need for sensitive and efficient detection methods. In this research, a novel spectrophotometric method utilizing boron-doped carbon quantum dots (<b>B-CQD</b>s) was developed for the detection of putrescine in artificial blood samples. B-CQDs, being a promising member of semiconductor quantum dots, offer improved optical properties along with enhanced biocompatibility and stability compared to conventional quantum dots. B-CQDs were synthesized <i>via</i> a straightforward method using boric acid, citric acid, and urea. The B-CQDs were characterized by Fourier-transform infrared spectroscopy, scanning electron microscopy, dynamic light scattering, and ultraviolet-visible spectroscopy, and the absorbance wavelength of B-CQDs was found to be 204.0 nm. The addition of putrescine to B-CQDs resulted in an increase in absorbance, enabling the quantification of putrescine through absorbance difference measurements. Optimization studies revealed that the developed method exhibits maximum sensitivity at pH 6.0, within the stable putrescine concentration range (1.0 µM). The linear working range of the method was found to be 0.05–0.75 µM with a high correlation coefficient (<i>R</i>² = 0.9975). The stability of the B-CQDs solution was evaluated, indicating its suitability for at least 20 min of continuous measurements. The applicability of the developed method was validated by analyzing putrescine in artificial blood samples, yielding satisfactory recovery values (89.5 ± 0.9% and 98.3 ± 0.6% for two different concentrations).</p>