Objective To observe the effect of ultrasound probe fixation bracket assisted ultrasound-guided venipuncture in difficult intravenous access. Methods A total of 105 inpatients with difficult intravenous access from November 2017 to May 2019 were selected by convenient sampling method. From November 2017 to May 2018, 39 patients as the control group were treated with ultrasound-guided peripheral venous indwelling needle puncture technique. From June 2018 to May 2019, 66 patients as the experimental group were treated with ultrasound probe fixation bracket assisted ultrasound-guided peripheral vein indwelling needle puncture technique. The success rate of one needle puncture, the times of puncture and the degree of pain were compared between the two groups. Results The success rate of one needle puncture was 92.4% (61/66) in the experimental group and 66.7% (26/39) in the control group (χ2 value was 11.450, P<0.01); The puncture times in the experimental group and the control group were 1.08±0.27 and 1.44 ±0.72, and the difference was statistically significant (Z value was -3.435, P<0.01). The pain scores of the experimental group and the control group were 2.00 ± 0.95 and 3.08±1.37, and the difference was statistically significant (Z value was -4.159, P<0.01). Conclusions The application of ultrasound probe fixation bracket assisted ultrasound-guided peripheral venipuncture technique in patients with difficult intravenous access can improve the success rate of one needle puncture, reduce the number of puncture and reduce the degree of pain. Key words: Ultrasound-guided; Peripheral vein; Indwelling needle; Vascular grading; Difficult intravenous access