Eastern equine encephalitis virus (EEEV) usually cycles between Culiseta melanura mosquitoes and birds; however, it can also infect humans. EEEV has a positive-sense RNA genome that, in infected cells, serves as an mRNA for the P1234 polyprotein. P1234 undergoes a series of precise cleavage events producing four nonstructural proteins (nsP1-4) representing subunits of the RNA replicase. Here, we report the construction and properties of a trans-replicase for EEEV. The template RNA of EEEV was shown to be replicated by replicases of diverse alphaviruses. The EEEV replicase, on the other hand, demonstrated limited ability in replicating template RNAs originating from alphaviruses of the Semliki Forest virus complex. The replicase of EEEV was also successfully reconstructed from P123 and nsP4 components. The ability of EEEV P123 to form functional RNA replicases with heterologous nsP4s was more efficient using EEEV template RNA than heterologous alphavirus template RNA. This finding indicates that unlike with previously studied Semliki Forest complex alphaviruses, P123 and/or its processing products have a leading role in EEEV template RNA recognition. Infection of HEK293T cells harboring the EEEV template RNA with EEEV or Western equine encephalitis virus prominently activated expression of a reporter encoded in the template RNA; the effect was much smaller for infection with other alphaviruses and not detectable upon flavivirus infection. At the same time, EEEV infection resulted only in a limited activation of the template RNA of chikungunya virus. Thus, cells harboring reporter-carrying template RNAs can be used as sensitive and selective biosensors for different alphaviruses. IMPORTANCE Infection of EEEV in humans can cause serious neurologic disease with an approximately 30% fatality rate. Although human infections are rare, a record-breaking number was documented in 2019. The replication of EEEV has a unique requirement for host factors but is poorly studied, partly because the virus requires biosafety level 3 facilities which can limit the scope of experiments; at the same time, these studies are crucial for developing antiviral approaches. The EEEV trans-replicase developed here contributes significantly to research on EEEV, providing a safe and versatile tool for studying the virus RNA replication. Using this system, the compatibility of EEEV replicase components with counterparts from other alphaviruses was analyzed. The obtained data can be used to develop unique biosensors that provide alternative methods for detection, identification, quantitation, and neutralization of viable alphaviruses that are compatible with high throughput, semiautomated approaches.