The invention provides a liquid crystal droplet and a preparation method thereof, a biosensor and a method for detecting a biological sample. The preparation method comprises the following steps: mixing a liquid crystal molecule with an amphiphilic molecule to form a mixture; and putting an ultrasonic wave transmission source into the mixture, and directly performing ultrasonic emulsification treatment on the mixture so as to obtain the liquid crystal droplet. Therefore, the liquid crystal molecule and the amphiphilic molecule can be effectively assembled, the obtained liquid crystal droplet has large number and good uniformity, and a substance which can be combined with amphiphilic molecules in the environment of the liquid crystal droplet can be quickly, sensitively and efficiently detected.