It's not true.
For simplicity, suppose $A$ is unital, so that its spectrum is compact Hausdorff.
If $X$ is a compact Hausdorff space and $C(X)$ is separable, then you can show that $X$ is second countable. (Let $\{f_k\}$ be a countable dense subset of $C(X)$, and $\{U_n\}$ a countable basis of open sets in $\mathbb{C}$; then the countable collection of sets $\{f_k^{-1}(U_n)\}$ is a basis for $X$, using Urysohn's lemma.) In particular, such an $X$ would be metrizable (by the Urysohn metrization theorem).
So the forward direction of your claim is true, since a second countable space is always separable, but this also suggests how to find a counterexample to the reverse direction: take a compact Hausdorff $X$ which is separable but not metrizable. One standard example is $X = [0,1]^{[0,1]}$ with its product topology; considering it as the space of all functions from $[0,1]$ to itself, the set of polynomials with rational coefficients is dense. Another, as YCor and Jochen Wengenroth discussed, is $X = \beta \mathbb{N}$, the Stone-Cech compactification of $\mathbb{N}$.
The converse of the above statement is also true, as user131781 points out: if $X$ is a compact metric space, then $C(X)$ is separable.
So the correct version of your claim (including the non-unital case) would be that $A$ is separable iff $\Omega(A)$ is second countable iff $\Omega(A)$ is a separable metrizable space.
$\Omega(A) :=$ {$\chi : A \to \mathbb C$ : $\chi$ is algebra homomorphism $\neq 0$}
but $\Omega(A) \mathrel{:=} \{\chi : A \to \mathbb C \mathrel: \text{$\chi$ is algebra homomorphism $\ne0$}\}$$\Omega(A) := \{\chi : A \to \mathbb C : \text{$\chi$ is algebra homomorphism $\neq 0$}\}$
. I have edited accordingly. Also, what do you mean by "the closure of any separable basis on $\Omega(A)$"? Basis is coll'n of open sets, right? $\endgroup$