The invention discloses a surface plasma resonance sensing chip and a preparation method and application thereof. The preparation method comprises the steps that firstly, a glass substrate is washed and then dried; secondly, a chromium film is prepared, a gold film is prepared on the chromium film, and a naked gold chip is obtained; thirdly, a solution containing modified bovine serum albumin is dropwise added, the whole chip is paved with the solution, the solution continues to be dropwise added until the whole chip is soaked, and after a sealed vibration reaction is carried out for 0.5-2 hours, washing and drying are carried out; fourthly, a mixed solution of NHS and EDC is dropwise added, the whole chip is paved with the mixed solution, the solution continues to be dropwise added until the whole chip is soaked, and after a sealed vibration reaction is carried out for 15 minutes to 1 hour, washing and drying are carried out; fifthly, a bromoacetic acid solution is dropwise added, the whole chip is paved with the bromoacetic acid solution, the bromoacetic acid solution continues to be added until the whole chip is soaked with the bromoacetic acid solution, after a sealed vibration reaction is carried out for 8-24 hours, washing and drying are carried out, and a blank chip is obtained. According to the preparation method, the surface plasma resonance sensing chip can be obtained within only 2 days and has better homogeneity than a traditional glucan chip which is prepared within 5 days.