Significant size heterogeneity in barramundi Lates calcarifer juveniles has been frequently reported in hatchery practices. Huge variation of mortality in different size grade of juvenile fish has also been observed. In order to explore the effect of size heterogeneity on gene expression level of barramundi juveniles, we graded them according to body length and selected growth-related genes (GHRH, GH, GHR, IGF-I, IGF-II, MSTN-1 and MSTN-2), and immune-related genes (IL8, IL10, TGFβ1, TNF, INF-γ1, Mx, CRP, C3, C4, mTOR, mLST8, eIF4E, HSP70 and HSP90) to measure their relative expression level. Results from the present study indicate that the expression level of GHR, IGF-I and IGF-II is consistent with body length, but there is no similar rule between GHRH, GH, mstn-1 and mstn-2. TOR pathway related genes were more actively expressed in L grade. Heat shock protein has no obvious rule in body length grade. L grade pro-inflammatory cytokines were up-regulated, but anti-inflammatory cytokines were down-regulated and inflammatory markers were up-regulated, indicating an obvious inflammatory response. The expression levels of S and M grade anti-inflammatory and proinflammatory cytokines was more balanced. This study provides the basis for finding the relationship between size heterogeneity and gene expression level of barramundi juveniles, and may provide insights for further understanding on the causes of size heterogeneity.