Abstract: A 80-day feeding trial was conducted to assess the interactive effects of β-glucan (BG) and Heat-killed Lactobacillus plantarum (HK-LP) on growth performance, intestinal fatty acid composition, and immune function of juvenile loach Misgurnus anguillicaudatus. Fish average initial weight of (0.17±0.01) g were fed with six experimental formulated diets containing two levels of BG (0 and 1%) with three levels of HK-LP (0.025%, 0.05%, and 0.1%) in triplicate. Results showed that supplementation of BG and HK-LP increased the body weight gain, specific growth rate, while significantly reduced the feed conversion ratio of fish. However, interaction between BG and HK-LP showed no significant effect on those parameters. Supplementation of BG remarkably decreased the percentage of C16鲶1n-7 and C18鲶2n-6 in intestinal fatty acid composition. Meanwhile, the percentage of C22 鲶1n-11 was decreased by dietary HK-LP. Dietary BG, HK-LP and two-factor interactions significantly influenced the activities of alkaline phosphatase (AKP) from skin mucus. Increase of dietary BG and HK-LP levels enhanced the activities of lysozyme. Supplementation of dietary 1% BG and 0.05% HK-LP significantly increased the expression levels of Hsp70 and Hsp90α. Therefore, the addition of BG and HK-LP in the diets could improve the growth performance of juvenile loach, and in the experiment, the optimum level of BG and HK-LP in the loach diet was 1% and 0.1%, respectively.