A novel sensing material for L-hydroxyproline (Hyp) detection was developed by synthesizing a bismuth film (BiF) and poly(L-hydroxyproline) (Poly(Hyp)) on a screen-printed graphene electrode (SPGE). Initially, the BiF electrodeposition was produced on the SPGE surface, followed by the Poly(Hyp) electropolymerization, resulting in the obtained Poly(Hyp)/BiF/SPGE. The morphology of a sponge-like thin film of Poly(Hyp)/BiF on SPGE had uniform nanometer-sized cavities on the graphene surface, offering a large electroactive area for interaction with the target substance. Cyclic and differential pulse voltammetry was used to examine the sensing performance of the proposed sensor, which revealed that the Poly(Hyp)/BiF/SPGE had the highest response toward Hyp detection. This was attributed to BiF and Poly(Hyp) can facilitate the transfer of electrons at an electrode/solution interface, resulting in an effective sensor for the detection of Hyp. Under the optimal conditions, the quantification of the proposed sensor was found to be linearly related to Hyp concentrations in the range of 0.01–5.0 mM with a limit of detection of 9.2 μM. Moreover, the interference of other substances detected in biological fluids showed no effect based on the ±5% error, indicating good selectivity for Hyp detection. In real applications, the proposed assay successfully analyzed Hyp in human urine samples, yielding satisfactory results with recoveries in the acceptable range of 98%–102%. Therefore, this facilely synthesized approach could be a suitable candidate to obtain a material to fabricate a new sensor for the measurement of Hyp, an important biomarker in the human body.