Background: Evidence is growing regarding the ability of platelet-rich plasma (PRP) injections to enhance functional capacity and alleviate pain in knee osteoarthritis (OA). However, heterogeneity in common practice regarding PRP preparation and biological content makes the initiation of this activity in a hospital complex. The aim of this study was to document the efficacy of a single PRP injection to treat knee OA and validate a routine care procedure. Methods: Fifty-seven patients with symptomatic knee OA received a single injection of large volume of very pure PRP. They were assessed at baseline and after one, three and six months, by measuring Knee Injury and Osteoarthritis Score (KOOS), Observed Pain after a 50-foot walk test and Visual Analog Scale (VAS) assessments. Magnetic Resonance Imaging (MRI) analysis was performed at baseline and six months after the procedure. The objective was to recover 50% of responders three months after the procedure using OMERACT-OARSI criteria. Results: A single administration of high volume pure PRP provided significant clinical benefit for 84.2% of the responders, three months after the procedure. The KOOS total score significantly increased from 43.5 ± 14.3 to 66.4 ± 21.7 six months after the procedure (p < 0.001). Pain also significantly decreased from 37.5 ± 25.1 to 12.9 ± 20.9 (p < 0.001). No difference was observed on MRI parameters. Conclusion: A single injection of large volume of very pure PRP is associated with significant functional improvement and pain relief, allowing initiation of daily PRP injection within our hospital.