Background: The treatment landscape of hepatocellular carcinoma (HCC) evolves, but the optimal sequential strategy for tyrosine kinase inhibitor (TKI) and immune checkpoint inhibitor (ICI)-based combination therapy is still unclear. This study compared the outcome and efficacy of TKI-(TKI+ICI) sequential therapy versus first-line TKI+ICI combination therapy in unresectable HCC. Methods: The patients who had received TKI (lenvatinib or sorafenib) plus ICI (anti-PD-1/PD-L1 monoclonal antibody) as first-line therapy or TKI-(TKI+ICI) sequential therapy were retrospectively included. Treatment efficacy (progression-free survival (PFS) and overall survival (OS)) to sequential therapy or TKI+ICI combination therapy as well as treatment-related adverse events (TRAEs) were assessed. Results: Among 392 patients included, 55 underwent TKI-(TKI+ICI) sequential therapy and 337 underwent first-line TKI+ICI combination therapy. The median PFS was significantly longer in TKI-(TKI+ICI) group than that in TKI+ICI group (16.2 months versus 7.7 months, HR 0.65 [0.47-0.90]; p=0.009). The median OS was not reached in TKI-(TKI+ICI) group and was 22.8 months in TKI+ICI group (HR 0.53 [0.33-0.83]; p=0.006). Multivariate analyses revealed that Child- Pugh score, BCLC stage and sequential therapy were independently associated with PFS. Child-pugh score, macrovascular invasion (MVI), AFP levels and sequential therapy were independently associated with OS. Subgroup analysis indicated that patients with younger age, male gender, an ECOG PS 1, Child-Pugh score A, HBV(+), HCV(-), BCLC stage C, presence of MVI or extrahepatic metastasis could remarkably benefit from sequential therapy. The survival outcome and objective response to TKI+ICI combination therapy was comparable between groups. The incidence of TRAEs were slightly higher in TKI-(TKI+ICI) group (p=0.022), but the difference was not significant for Grade≥3 events (p=0.126). Conclusions: TKI-(TKI+ICI) sequential therapy is a valid therapeutic strategy for patients with HCC compared with first-line TKI+ICI combination therapy.